CN107375920A

CN107375920A - A kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use

Info

Publication number: CN107375920A
Application number: CN201710486697.1A
Authority: CN
Inventors: 吕宏亮; 董金杰; 张�杰; 田波; 邓瑞雪; 王会保; 刘萍; 刘西兰; 景志忠; 高世杰; 王超英; 张云德; 张永光; 殷宏
Original assignee: CHINA AGRICULTURAL VET BIO SCIENCE AND TECHNOLOGY Co Ltd
Current assignee: CHINA AGRICULTURAL VET BIO SCIENCE AND TECHNOLOGY Co Ltd
Priority date: 2017-06-23
Filing date: 2017-06-23
Publication date: 2017-11-24
Anticipated expiration: 2037-06-23
Also published as: CN107375920B

Abstract

The invention discloses a kind of porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virosomes vaccine and its production and use.The vaccine contains porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct subvirion antigens and stablizes adjuvant compound, the porcine reproductive and respiratory syndrome virus swine influenza virus reconstruct virion is to be formed by porcine reproductive and respiratory syndrome virus by directly adsorbing or covalentlying bind in swine influenza virus reconstruct virosomal surface, wherein, the swine influenza virus reconstruct virion is the small vesica containing phospholipid bilayer, and swine influenza virus hemagglutinin and neuraminidase protein are combined with phospholipid bilayer layer surface.The vaccine that the present invention is prepared has the characteristics that safe efficient, easy to use, the effect phase is long.Reduction is can reach by Nasal immunization and/or removes breath syndrome virus and/or swine influenza virus purpose.

Description

A kind of porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virosomes vaccine And its production and use

Technical field

The present invention relates to a kind of porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virosomes vaccine and its preparation Method and purposes.The invention belongs to biological medicine or veterinary biological product technical field.

Background technology

Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) be pig chronic disease, it is popular in the country of whole world cultivation pig, cause economic loss about 40,000,000,000 every year, be pig cultivation and meat One of most important infectious disease of class industry.

From PRRSV virus find since, develop both at home and abroad inactivation and attenuation modification live vaccine (Modified live, PRRS-MLV) it is used to PRRSV control.Nevertheless, PRRS-MLV has to susceptible pig recombinates variation risk, live vaccine strain is also poisonous The possibility of power reversion is low to gm allotype virus attack level of protection, it is impossible to eliminate virus infection or persistent infection, toxin expelling.Go out Although live vaccine security is good, immunogenicity is weak, and immune effect is bad.Therefore, be badly in need of developing it is a kind of safe efficient and The non-infectious PRRSV vaccines to viral cross protection can be realized.

Swine flu infection causes the huge economic loss of pig industry, be public health and the maximum disease of pig industry harm it One.Swine flu is a kind of acute respiratory disease caused by influenza A virus (Swine influenza A virus, SIAV) Disease, it is related to schneiderian membrane, tonsillotome, tracheae, pulmonary epithelial cells.Major Epidemic H1N1, H1N2, H3N2 swine flus in swinery at present Alphavirus.Swine influenza virus genome is the strand RNA of 8 fragments, encodes 12-13 albumen：PB2、PB1、PB1-F2、PB1- N40、PA、PA-X、HA、NP、NA、M1、M2、NS1、 NS2/NEP.Swine influenza virus infection causes pig high fever, drowsiness, appetite to subtract Move back, have difficulty in breathing, cough.Although disease continue 2-6 days, the death rate it is low, most pigs recover, and body weight seriously reduces.Swine flu with Other respiratory pathogenses coinfections cause pig chronic respiratory disease and compound respiratory disease syndrome, and swine influenza virus infection sow is accidental Cause miscarriage.It is some of because pig is people, influenza virus A avian blender, therefore can produce many different SIAV strains With the potentiality infected between humans and animals.The effective immune pig industrial economy that can prevent loses, limits pig Flu-A Virus is propagated to people.Although bivalent or multivalence intact virus inactivated vaccine can protect homologous virus infection, to heterologous strain Protect limited.

In vaccine manufacture, often containing animal derived albumen, such as ox or human serum albumins, animal in vaccine formulation Gelatin is with enhanced virus class vaccine, the stability of particularly environmentally sensitive virus type vaccine, but the albumen in vaccine component If not having controlled pig, ox or horse from zoonosis, the potential risk for having infectious disease transmission, also have and cause metamorphosis Reaction or the risk of animal stress reaction.Therefore need to develop a kind of excipient without animal protein, polypeptide and oligopeptides of stabilization Agent, for protecting the stability and integrality of vaccine antigen, totivirus is purified comprising inactivation especially for be difficult to preserve Pig breeds and Respirovirus vaccine and swine flu vaccine；It is also required to develop the epidemic disease that suitable effective dose is low, total protein content is small The excipients that seedling uses.Highly purified vaccine antigen, because being excluded in vaccine formulation containing micro residual impurity albumen and nucleic acid Potential allergic reaction, but then can cause the degraded of purifying inactivation of viruses, absorption or cohesion so that vaccine activity into Reduction, effect is divided to reduce, therefore needed in excipients containing adjuvant and antigen stabilizer is stablized, the single aluminium of vaccine is stable at present helps Agent, the stable adjuvant of two-way oil are difficult the immunogenicity and security for keeping inactivated vaccine, it is therefore necessary to which exploitation can make vaccine The effect phase is long and can induce the stable adjuvant of specific humoral immunity and cellular immunity.

The content of the invention

The problem of existing for the existing vaccine of current porcine reproductive and respiratory syndrome virus, it is an object of the invention to use Virus culture, purification technique, viral Antibody Production Techniques, there is provided one kind is safe efficient, easy to use, the effect phase is long and can realize The collunarium vaccine protected to PRRSV and swine influenza virus infection.

In order to achieve the above object, present invention employs following technological means：

A kind of porcine reproductive and respiratory syndrome virus of the present invention-swine influenza virus reconstruct virosomes vaccine, it is numerous containing pig Grow and answered with breathing syndrome virus-swine influenza virus reconstruct subvirion antigens (PRRSKPWV-SwIVA-RV) and stable adjuvant Compound, described porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct subvirion antigens are comprehensive with breathing by pig breeding Close what syndrome virus was formed by directly adsorbing or covalentlying bind in the surface of swine influenza virus reconstruct virion, wherein, it is described Swine influenza virus reconstruct virion be the vesicles containing phospholipid bilayer, be combined with pig on the surface of phospholipid bilayer Influenza virus hemagglutinin (haemagglutinin, HA) and neuraminidase (neuraminidase, NA) albumen.

In the present invention, it is preferred to, described stable adjuvant compound is must be with nonessential ammonia containing 2.0~10g/L Base acid blend, 20~25g/L poly-D-lysine hydroxymethyl celluloses compound (Poly ICLC), 0.1-0.5mg/L cards are situated between Bacterium lysate, 30~100g/L disaccharide, 30~100g/L polyalcohols, 1.0~5.0g/L urea or urea derivative, 0.01~ 0.4g/L EDTA or edta salt, 0.001~0.1g/L surfactants 10~100mM cushioning liquid, pH value 7.0~ 9.0, more preferably pH 7.2-8.0.

The present invention stable adjuvant complex components meet pharmacopeia, veterinary drug allusion quotation, veterinary science, medical requirement, available for people with And the preparation of the vaccine of animal.The composition of the particularly stable adjuvant of vaccine can not contain the virus of interference infringement vaccine antigen or work Composition, play gathering for BCG vaccine lysate and poly-D-lysine hydroxymethyl cellulose for adjuvant effect in stable adjuvant compound Flesh born of the same parents.Antibiotic, the content of divalence salt ion, concentration are all permitted for beast pharmacopoeial requirements in stable adjuvant compound, and without pair The harmful composition of animal, human body and food security, these compositions are clinical for many years into the pharmacology after body, toxicological effect Confirmed in use.Illustrate that vaccine ensures the security of vaccine from the selection of raw and auxiliary material.

The poly-D-lysine hydroxymethyl cellulose compound with adjuvant effect is in the stable adjuvant compound of the present invention The effective, derivant of pharmaceutical grade safe to use.Used in some treatments of cancer of people and as antiviral drugs, safety.It is more Polylysine hydroxymethyl cellulose compound induction different animals cell produces I interferoids performance antivirus action and is immunized and adjusts Section acts on and the effect of inducing specific antibody tormation.Particulate antigen plays it with the vaccine for stablizing the preparation of adjuvant compound and exempted from The facilitation and acted in intranasal inducing interferon topical antiviral that epidemic disease effect regulation, specific antibody generate.

People mainly plays the facilitation of cellular immunity and humoral immunity with the protein lysates of BCG vaccine, can make breathing Mucous membrane absorbs to PRRSKPWV-SwIVA-RV, submission strengthens, and so as to strengthen part and systemic immunity reaction, makes vaccine immunity Phase extends, immune effect significantly improves, and expands to different genotype, the immune spectrum of different genetic pedigree PRRSV, SwIVA.Also It is safety, wide spectrum, the high efficiency for improving invention vaccine.

Contain nonionic surfactant in the stable adjuvant compound of the present invention, PRRSKPWV-SwIVA- can be made RV immunogenicities or biological activity are stable, and virion keeps complete, prevents PRRSKPWV-SwIVA-RV from adsorbing in chamber wall Upper, prevention PRRSKPWV-SwIVA-RV condenses, degrades or combined.

Nonionic surfactant makes in vaccine combination composition poly-D-lysine hydroxymethyl cellulose compound in various bars Stable not mutability is annealed under part, is not easy to lose its activity also guarantee PRRSKPWV-SwIVA-RV antigens stabilization.Contribute to it to enter Enter the effect that body cell plays inducing endogenous interferon, play innate immune and adoptive immunity effect.

The pH value of the stable adjuvant compound of the present invention is in 7.0-9.0, according to PRRSV, SwIVA physicochemical property, it is preferred that The more suitable virus activities of pH of buffer 7.2-8.0 and structural integrity.It is steady in this scope PRRSKPWV-SwIVA-RV particles It is qualitative to protect well, it is especially relatively stable to porcine reproductive and respiratory syndrome virus.

In the present invention, it is preferred to, described must comprise at least arginine or smart ammonia with non-essential amino acid blend Hydrochlorate and glutamic acid or glutamate；Described disaccharide is disaccharide, preferably maltose, fucose or sucrose wherein at least one Kind, described polyalcohol be polyalcohol, preferably D-sorbite or mannitol or its combination, described surfactant be it is non-from Sub- surfactant, preferably Tween-20 or Pluronic F68；Described cushioning liquid is selected from phosphate buffer, Tris is buffered The mixture that any one or more than one of liquid or HEPES buffer solution are formed in arbitrary volume ratio.

In the present invention, it is preferred to, described BCG vaccine lysate is prepared in accordance with the following methods：BCG vaccine is logical with improvement Soviet Union 37 DEG C of synthetic medium static gas wave refrigerator 2-3 weeks, culture at 121 DEG C 30 minutes carry out sterilization processing, with preparative low speed from Scheming harvest thalline, PBS wash 2 times, be suspended in containing EDTA, protease inhibitors, DNA enzymatic, RNase PBS in, use glass Pearl crushes bacterium, until 90% bacterial cell disruption, the unbroken cell of centrifugation, insoluble cell wall constituent, harvests in cracking Clear liquid；Supernatant obtains whole cell lysate, wherein level of endotoxin should be not higher than through 0.2 μM, low protein bound membrane filtration 0.002 μ g/mg albumen.

In the present invention, it is preferred to, in described vaccine, porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct The content of virion is 4-8 μ g/ml.

Further, the invention also provides one kind prepares the porcine reproductive and respiratory syndrome virus-swine influenza virus Virosomes vaccine method is reconstructed, is comprised the following steps：

(1) preparation of the PRRSV totivirus antigens of purifying inactivation；

(2) preparation of the swine influenza virus totivirus antigen of purifying inactivation；

(3) preparation of swine influenza virus reconstruct virion

The preparation of a phospholipid dispersions

Prepared with the 0.01M Tris/HCl containing pH 7.3,0.1M NaCl in homogenizer and contain phosphatide and cholesterol The dispersion liquid of mixture, wherein containing 75% phosphorus in percentage by weight in described phosphatide and the mixture of cholesterol Phosphatidylcholine, 20% phosphatidyl-ethanolamine and 5% cholesterol, wherein all phosphatide account for the 1-2% (w/v) of dispersion liquid, Sodium taurocholate is added in dispersion liquid, makes its final concentration of 1.3% (w/v)；

The preparation of b swine influenza virus outer membrane proteins

Added into the swine influenza virus virus liquid of purifying containing the glycol list dodecyl ethers of 0.1M eight, 7.9mg/ml The pH 7.3 of NaCl, 4.4mg/ml sodium citrate, 2.1mg/ml MES, 1.2mg/ml N- ethoxys-piperazine-N'-2- ethyl sulfonic acids The aqueous solution, mixture centrifuges in ultracentrifuge, collects supernatant, obtain viral envelope proteins, i.e. hemagglutinin (HA) and god Through propylhomoserin enzyme (NA)；

C swine influenza viruses reconstruct the preparation of virion

The supernatant that step b is obtained is added in the phospholipid dispersions that step a is obtained, in 4 DEG C of stirrings, loading Sephadex G-50 chromatographic columns, pillar is placed to be connected with Ultrasound Instrument in a water bath, and ultrasonic vibration per minute produces swine flu in 10 seconds Virus reconstruct virion, reconstituted influenza virion and cholesterol micro-capsule flow out in outer water section, collect void volume part, will contain The outer hydration of swine influenza virus reconstructed volume simultaneously, chromatographs again under similarity condition, obtains swine influenza virus reconstructed volume；

(4) coupling of porcine reproductive and respiratory syndrome virus and swine influenza virus reconstruct virion

The swine influenza virus that the PRRSV totivirus antigen and step (2) that the inactivation that step (1) is obtained purifies obtain reconstructs Body mixing, resuspension is gently shaken, being gently mixed at 20 DEG C 48 hours makes PRRSV be adsorbed in swine influenza virus weight by Van der Waals force The surface of structure virion, obtain porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virion；Or

Disulfide bond be present using PRRSV surfaces and carried out covalent bond virion with swine influenza virus reconstruct virion.

Further, described porcine reproductive and respiratory syndrome virus-swine flu is prepared the invention also provides another Virus reconstruct virosomes vaccine method, comprises the following steps：

(1) preparation of the PRRSV totivirus antigens of purifying inactivation

(2) PRRSV's is Thiolation

The PRRSV of inactivation purifying is dissolved in 0.1M phosphate buffers；N- succinimido pyridine radicals dithiopropionic acid esters (SPDP) mixed with ethanol, take mixed liquor to be slowly added to the phosphate buffer containing PRRSV with syringe, make SPDP and PRRSV Mol ratio 15:1, while keep concentration of alcohol to prevent albuminous degeneration in below 5v/v%, mixture reacts 30 minutes at 20 DEG C, Reaction terminate after, respectively with pH7.0 0.05M sodium citrates, pH7.0 0.05M sodium phosphates, pH7.0 0.05M sodium chloride Balance Sephadex G-50 pillar is purified three times；Obtain Thiolation PRRSV totivirus antigens PRRSV-SPDP；

(3) preparation of the swine influenza virus totivirus antigen of purifying inactivation

(4) preparation of swine influenza virus reconstruct virion

The preparation of a phospholipid dispersions

Phosphatidyl choline (PE) and N- succinimido pyridine radicals dithiopropionic acid esters (SPDP) are crosslinked：15mg Phosphatidyl choline is put into the drying of 5ml vials, is redissolved in after drying in chloroform, then adds containing triethylamine (TEA), SPDP 1-2 hours are stirred at room temperature until reaction completion, i.e., no single phosphatidyl courage in absolute ethyl alcohol, mixture under logical condition of nitrogen gas Alkali, reaction product are dried on a rotary evaporator, and product is resuspended in chloroform, and loading silicic acid chromatography post is purified, and obtains PE- SPDP；

Prepared with the 0.01M Tris/HCl containing pH 7.3,0.1M NaCl in homogenizer and contain phosphatide and cholesterol The dispersion liquid of mixture, wherein containing 75% in percentage by weight in described phosphatide and the mixture of cholesterol PE-SPDP, 20% phosphatidyl-ethanolamine and 5% cholesterol, wherein all phosphatide account for the 1-2% (w/v) of dispersion liquid, will Sodium taurocholate is added in dispersion liquid, makes its final concentration of 1.3% (w/v)；

The preparation of b swine influenza virus outer membrane proteins

C swine influenza viruses reconstruct the preparation of virion

(5) coupling of porcine reproductive and respiratory syndrome virus and swine influenza virus reconstruct virion

By Thiolation PRRSV totivirus antigen PRRSV-SPDP pH 1M in 0.2M citrate phosphate buffers HCl is adjusted to 5.5, adds dithiothreitol (DTT) solution, and solution is placed 30 minutes, and Sephadex G-50 separation is balanced with PBS Dithiothreitol (DTT), albumen is collected in having nitrogen environment, the Thiolation PRRSV totivirus antigen after being reduced；By acquisition Reconstruct swine influenza virus body and reduction after Thiolation PRRSV totivirus antigen be stirred at room temperature overnight, obtain pig breeding with Breathing syndrome virus-swine influenza virus reconstituted virosomes.

In method described above, the preparation of the PRRSV totivirus antigens of described purifying inactivation comprises the following steps：

(1) Virus culture, three times freeze-thaw, harvest

Porcine reproductive and respiratory syndrome virus infection MARC-145 cells, harvest virus-culturing fluid, collect PRRSV viruses Liquid is through freeze-thaw three times, and lysate centrifugation, supernatant collection is in sterile chamber tank；

(2) inactivate

The PRRSV virus liquids for taking step (1) to obtain add inactivator beta-propiolactone, obtain PRRSV inactivation of virus liquid；

(3) ion-exchange chromatography

Virus liquid after inactivation is chromatographed with ion exchange column；

(4) it is concentrated by ultrafiltration, dialyses

With cutoff value it is that 300KD films are concentrated by ultrafiltration from the virus liquid of ion exchange column elution, while with 0.02 M Tris-HCl (pH 7.5) dialyses；

(5) molecular exclusion chromatography

The chromatographic columns of PRRSV virus liquid loadings FF-Sepharose 6 are concentrated, obtain viral purification liquid；

(6) it is concentrated by ultrafiltration, dialyses

Viral purification liquid is used into film of the cutoff value for 300KDa, using containing 150mM NaCl, pH 7.5 50mM phosphorus Acid buffer ultrafiltration dialysis, obtain inactivation purifying PRRSV totivirus liquid.

In method described above, the preparation of the swine influenza virus totivirus antigen of described purifying inactivation is including following Step：

(1) Virus culture, three times freeze-thaw, harvest

Swine influenza virus infection mdck cell, virus-culturing fluid is harvested, collects swine influenza virus virus liquid through freeze-thaw three times, Lysate centrifuges, and supernatant collection is in sterile chamber tank；

(2) inactivate

The swine influenza virus virus liquid for taking step (1) to obtain adds inactivator formalin, obtains swine influenza virus inactivation Liquid；

(3) ion-exchange chromatography

Virus liquid after inactivation is chromatographed with ion exchange column；

(4) it is concentrated by ultrafiltration, dialyses

With cutoff value it is that 700KD films are concentrated by ultrafiltration from the virus liquid of ion exchange column elution, while uses 0.02M Tris-HCl (pH 7.5) dialyses；

(5) molecular exclusion chromatography

The chromatographic columns of swine influenza virus virus liquid loading FF-Sepharose 6 are concentrated, obtain viral purification liquid；

(6) it is concentrated by ultrafiltration, dialyses

Viral purification liquid is used into film of the cutoff value for 700KDa, using containing 150mM NaCl, pH 7.5 50mM phosphorus Acid buffer ultrafiltration dialysis, obtain inactivation purifying swine influenza virus totivirus liquid.

Further, the invention also provides described porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct Virosomes vaccine is preparing the medicine of disease of the prevention caused by porcine reproductive and respiratory syndrome virus and/or swine influenza virus In purposes, it is preferred that described medicine is administered by way of collunarium.

In PRRSV vaccine preparation technologies, by veterinary biological product or medical biotechnology product requirement, advised as closing PRRSV vaccine antigen purification process is sucrose density gradient centrifugation, but time-consuming, purification effect and product purity are low, are difficult to again Amplification and scale use.As PRRSV purifying use heparin affinity chromatography, removal of impurity is but repeated it is reported that for 96% It is bad.To establish high-purity, high production capacity PRRS whole virus particles purifying process, the chromatographic technique group for meeting cGMP checkings has been invented The PRRSV particle purifying process of conjunction, the preparation for PRRSV vaccine antigens.It there is no at present using clarification, concentration, chromatography combination Technology purifies PRRSV report, and chromatographic technique combination meets cGMP certifications, makes on the virus type vaccine such as rabies With being more easy to be approved large-scale production.Compared with density gradient centrifugation, viral chromatographic purifying technique is quick, product consistency is good, easy In auto industrialization.In being commercialized, having met the chromatographic technique of cGMP certifications, molecular exclusion chromatography can remove PRRSV It is concentrated by ultrafiltration substantial amounts of impurity in liquid, the high conversion for being simultaneously easy to target formula buffer solution of yield, the step yield rate is up to 75%. After the clarified removal PRRSV stoste sheet cell fragments of PRRSV suspending nutrient solutions, further removed with ion-exchange chromatography big The nucleic acid of amount simultaneously concentrates PRRSV particles.Ion-exchange chromatography obtains PRRSV purity and obtained higher than sucrose density gradient centrifugation 2 times of PRRSV products.For this kind of big viruses of PRRSV, traditional Ion Exchange Medium is not suitable for this kind of big viruses of PRRSV Purifying.Technique, high yield are easily formed, is easily verified that, launch, commercially available production antigen or epidemic disease can be facilitated for a period of time The MEDIUM Q sepharose HP of seedling are as ion-exchange chromatography media used in the present invention：Unit volume adsorbs outer surface Product is big, therefore can provide preferable associativity.The step for saving the ultrafiltration concentration of traditional vaccine step needs simultaneously.

PH is the important parameter for developing ion-exchange step, influences PRRSV charge density.Albumen assembles strong depend-ence PH, therefore to avoid PRRSV from assembling and stably, liquid pH is selected and maintained 7.5.Another important parameter of ion-exchange chromatography For ionic strength, the interaction of PRRSV particle electrifications is influenceed, to maximize the acquisition of separating effect, preferably at most combines virus Ionic strength virus liquid loading ion exchange column to increase production capacity and washability.With reference to Q sepharoseHP posts linearly ladder Spend liquid elution, preferably equilibrium liquid NaCl containing 0.1M.Although after cation exchange chromatography, PRRSV purity improves, and its is anti- Answering property does not improve, and illustrates that purifying had not changed PRRSV structure.Chromatography buffer salinity influences PRRSV surface glycoproteins Glycosylation, the glycosylated membrane protein that salinity selected by invention is adapted to hold PRRSV is stable and do not influence the immunogenicity of vaccine. Ion-exchange chromatography is adapted to a large amount of DNA fragmentations or impurity removed in virus stock solution used.

Compared with existing conventional inactivated vaccine technology, the beneficial effects of the present invention are：

(1) inactivation purifying PRRS intact viruses and swine flu Alphavirus reconstruct virion (PRRSKPWV-SwIVA-RV) As vaccine antigen, vaccine is set to produce protective immune response to different genotype PRRSV and different subtype SwIVA infection；Can For a boar bigeminy vaccine, for preventing PRRSV, SwIVA infection, reduce or remove PRRSV viremia virusemias, reduce SwIVA senses Macroscopic view, microscopic damage, pathology caused by dye, reduce pig heating and clinical symptoms；

(2) antigen is PRRSKPWV-SwIVA-RV particles in vaccine of the present invention, is stablizing the effect of adjuvant compound Under, can be efficiently induction of Th1 immune responses, and general PRRSV or SwIVA inactivated vaccines only induce Th2 to react.Mean Invention vaccine can induce synthesis, the reaction of protectiveness humoral and cellular immune response.

(3) killed vaccine antigen commonly needs adjuvant enhancing immune response to make vaccine effective.The component of stable adjuvant Enhancement antigen individually caused immune response.In vaccine stabilizing agent of the present invention, further comprise one or more adjuvants, help Agent can be veterinary science and pharmaceutically acceptable combination.Preferably, described stable adjuvant component includes BCG vaccine strain whole cell egg White lysate and poly-D-lysine hydroxymethyl cellulose compound, make vaccine that there is tight security, immunity and good exempt from Epidemic disease effect.

(4) stable adjuvant compound and antigen manufacturing process of the invention are simple, cheap.

(5) vaccine of the present invention can strengthen the generation of neutralizing antibody through intranasal instil immune gilt, sow, farrowing sow, The PRRSV viremia virusemia duration is reduced, to isotype, gm allotype, the breeding of different genetic pedigree pigs and the comprehensive disease of breathing Poison infection causes symptom or death to have protective effect, has broad-spectrum immunogenicity；And safely its life is not influenceed to farrowing sow Grow, duration of immunity is 120 days.

(6) stable adjuvant compound of the invention makes PRRSKPWV-SwIVA-RV vaccines to be preserved at 2-8 DEG C 2 years, imitates the phase Up to 2 years.

(7) vaccine of the present invention provides intranasally inoculated mode, overcomes secondary caused by conventional inactivated vaccine intramuscular injection path Reaction, pig stress reaction is reduced, to pig without invasion, excite innate immune, adoptive immunity reaction.Safely, effectively, user Just.

Embodiment

The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention Row modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.

The preparation of the porcine reproductive and respiratory syndrome virus of embodiment 1-swine influenza virus reconstituted virosomes and vaccine

1st, strain, cell, nutrient solution

The present invention prepares vaccine and PRRSV antibody strain is highly pathogenic PRRSV HP- The 25th generation Strain HP-PRRSV-JXM-F25 that PRRSV-JXM-F5 strains are passed on Marc-145 cells.High-pathogenicity porcine Reproductive and respiratory syndrome virus HP-PRRSV-JXM-F5 strains, it is that HP-PRRSV-JXM was uploaded to for 5 generations in MARC-145 cells Adapted strain, Classification And Nomenclature is high-pathogenicity porcine reproductive and breathing syndrome virus, and its microbial preservation number is：CGMCC NO.9453, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date July 8 in 2014 Day, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.The Strain has been remembered It is on December 30th, 2014 to be loaded in the applying date, Application No. 201410842421.9, a kind of entitled " wide spectrum mucosal immunity In the patent application of the vaccine combination of prevention and control porcine reproductive and respiratory syndrome and its application ".

Without exogenous factor, meet PRRSV vaccine antigens production MARC-145 work storehouse cell be middle peasant Witter biology section Skill limited company builds storehouse and identification, is examined and determine for inventing the production of PRRSV vaccine antigens and external titre, biochemical analysis, carefully Born of the same parents' maintaining liquid (is purchased from the U.S. for the DMEM of calf serum containing 5v/v%, 100 μ g/ml streptomysins and 100IU/ml penicillin Invitogen companies) nutrient solution, virus-culturing fluid is containing 0.1% ox blood is pure or horse serum MEM nutrient solutions.

2nd, prepared by purifying inactivation PRRSV totivirus antigen

2.1 Virus cultures, three times freeze-thaw, harvest

MARC-145 cells are expanded with cell factory culture, are transferred to and are filled hyclone containing 5v/v%, 100 μ g/ml strepto-s Element and DMEM (Invitogen) nutrient solutions of 100IU/ml penicillin and microcarrier cytodex-3 bioreactor, volume 30L, effective working volume 25L, 37 DEG C, 5%CO₂Environment culture 3-4 days, nutrient solution pH7.2 ± 0.2, oxygen saturation 25 ± 0.1%, mixing speed middling speed.Porcine reproductive and respiratory syndrome virus HP-PRRSV-JXM-F25 Strain is infected with 0.01MOI MARC-145 cells, virus-culturing fluid was harvested at the 7th, 11,15 day respectively, is often harvested once, add virus liquid infection.Collect PRRSV virus liquids are through freeze-thaw three times, and lysate was in 4 DEG C of 5000g low speed continuous flow centrifugations 20 minutes, and supernatant collection is in sterile appearance Device tank.Amount to and obtain 5000 milliliters of PRRSV virus stock solution useds.

2.2 inactivation

The PRRSV virus liquids of above-mentioned acquisition are taken to add 1ml inactivator beta-propiolactone by 4000ml, in 22-25 DEG C of inactivation 18 hours, 37 DEG C hydrolyzed 2 hours, obtained PRRSV inactivation liquid.

Inactivate checking test：1mL inactivation liquid adds the 150cm equipped with 50mL nutrient solutions²Cell bottle, 37 DEG C are cultivated 1 week, are changed Fresh medium continues culture 1 week.The non-inactivation of viruses liquid for being inoculated with 1mL simultaneously observes cytopathic effect to same cell bottle.

2.3 ion-exchange chromatography

Added after virus liquid after inactivation is balanced with l20mM Tris-HCl (pH 7.5) buffer solution of the NaC containing 0.1M Ion exchange column (the Q sepharoseHP balanced through level pad (20mM Tris, 150mM NaCl, pH=7.5) Medium 100ml loads the chromatographic columns of XK 16/20, purchased from GE Healthcare companies), collection flows through liquid, with 10 times of column volumes Equilibration buffer solution, then successively with the 20mM containing 0.1M, 180mM, 490mM, 900mM NaCl of 10 times of column volumes Tris-HCl (pH 7.5) linear gradients liquid elutes, and collects each peak, detects OD₂₈₀Absorption value.

First eluting peak collection liquid is with after 10 times of 20mM Tris-HCl (pH 7.5) solution dilution of the NaCl containing 0.1M Sample ion exchange column, eluted with the incremental elution buffer of NaCl concentration, all step flow velocity 5ml/min.Collection contains The eluting peak of PRRSV particles and detection and analysis：Except 0.1M NaCl eluents, 180mM NaCl, 490 mM NaCl 900mM Equal particle containing PRRSV in NaCl eluents, the SDS-PAGE purity assays of 490mM NaCl eluents and sucrose density gradient from The heart or the PRRSV of heparin affinity chromatography post purifying are approached.Amount to the PRRSV virus liquids of 1000 milliliters of harvest.

2.4 are concentrated by ultrafiltration, dialysis

From ion exchange column elution virus liquid with cutoff value be 300KD films be concentrated by ultrafiltration 100 times, use simultaneously 0.02M Tris-HCl (pH 7.5) dialyse.

2.5 molecular exclusion chromatography

The chromatographic columns (being purchased from GE Healthcare companies) of 10ml concentration PRRSV virus stock solution used loadings FF-Sepharose 6. The equilibrium liquid of chromatographic column is 0.02M Tris-HCl (pH 7.5).Eluted with equilibrium liquid with 0.8ml/min flow velocitys, while detection is washed De- liquid is in OD₂₈₀Absorption value, collect elution first peak.First peak is the PRRSV of purifying.SDS-PAGE display inactivation concentrations PRRSV virus liquids eliminate a large amount of foreign proteins through molecular exclusion chromatography, and with PRRSV virus purifications.

Albumen contained by first peak is through SDS-PAGE electrophoresis showeds：Purify PRRSV viral N proteins molecular weight 15kDa, M albumen Molecular weight 18-19kDa, GP5 molecular weight of albumen 25kDa, GP2 molecular weight of albumen 29kDa, GP4 molecular weight of albumen 31kDa, GP3 egg White molecular weight is 42kDa.As a result show that molecular weight and the PRRSV major structural proteins of PRRSV purified products are in the same size.

Transmission electron microscope observing purifying PRRSV virus liquids observe a diameter of 25-30nm of kernel, diameter 50-74nm it is circular or Oval cyst membrane particle, also there are circle or oval cyst membrane particle of the sub-fraction diameter more than 60nm.Disease is obtained by the step Malicious 200 milliliters of refined solution.

2.6 are concentrated by ultrafiltration, dialysis

Viral purification liquid is used into film of the cutoff value for 300KDa, using phosphate buffer (50mM phosphate buffers, 150mM NaCl, pH 7.5) 2 times of dialysis are concentrated by ultrafiltration, 100 milliliters of inactivation purifying PRRSV totivirus liquid are obtained, for reconstructing disease The preparation of malicious particle.

3rd, the preparation and detection of swine influenza virus reconstruct virion

3.1 strain, cell, nutrient solution

Strain is swine influenza virus H1N2 strains (A/swine/LZ/001/2016), H1N1 (A/swine/GS/009/ 2017) voluntarily separate and identify for middle peasant Witter biotech inc.

Built without exogenous factor, the mdck cell for meeting vaccine antigen production for middle peasant Witter biotech inc Storehouse and identification, produce and examine and determine for invention vaccine antigen, cell maintenance medium is calf serum containing 5v/v%, 100 μ g/ml chains The DMEM of mycin and 100IU/ml penicillin (being purchased from Invitogen companies of the U.S.) nutrient solution, virus-culturing fluid are containing 0.1v/ V% ox blood is pure or horse serum MEM nutrient solutions, virus-culturing fluid (are purchased from U.S. Sigma- containing 3 μ g/ml TCPK trypsase Aldrich).

The preparation of the swine flu totivirus antigen of 3.2 purifying inactivations

3.2.1 Virus culture, three times freeze-thaw, harvest

Mdck cell with cell factory culture expand, be transferred to fill hyclone containing 5v/v%, 100 μ g/ml streptomysins with And DMEM (Invitogen) nutrient solutions of 100IU/ml penicillin and microcarrier cytodex 3 bioreactor, volume 30L, Effective working volume 25L, 37 DEG C, 5%CO₂Cultivated 3-4 days under environment, nutrient solution pH7.2 ± 0.2, oxygen saturation 25 ± 0.1%, mixing speed middling speed.Swine influenza virus H1N2 is infected with 0.01MOI, harvests Virus culture at the 7th, 11,15 day respectively Liquid, often harvest once, add virus liquid.In 4 DEG C of 5000g low speed continuous flow centrifugations 20 minutes, supernatant was hollow through 0.85 μm Fibrous filters carry out micro-filtration, and the virus liquid after micro-filtration is collected in sterile chamber tank.Amount to the swine influenza virus of 5000 milliliters of acquisition Stoste.

3.2.2 inactivation

Take above-mentioned acquisition swine influenza virus liquid 4000ml to add 1ml 5v/v% formalins, 48 are inactivated at 22-25 DEG C Hour, obtain swine influenza virus inactivation liquid.

3.2.3 ion-exchange chromatography

Added after virus liquid after inactivation is balanced with l20mM Tris-HCl (pH 7.5) buffer solution of the NaC containing 0.1M Ion exchange column (the Q sepharoseHP balanced through level pad (20mM Tris, 150mM NaCl, pH=7.5) Medium 100ml loads the chromatographic columns of XK 16/20, purchased from GE Healthcare companies), collection flows through liquid, with 10 times of column volume posts Equilibration buffer solution, then successively with 20mM Tris- of 10 times of column volumes containing 0.1M, 180mM, 490mM, 900mM NaCl HCl (pH 7.5) linear gradients liquid elutes, and collects each peak, detects OD₂₈₀Absorption value.

First eluting peak collection liquid is with after 10 times of 20mM Tris-HCl (pH 7.5) solution dilution of the NaCl containing 0.1M Sample ion exchange column, eluted with the incremental elution buffer of NaCl concentration, all step flow velocity 5ml/min.Collection contains pig The eluting peak of influenza virus particles and detection and analysis：Except 0.1M NaCl eluents, 180mM NaCl, 490mM NaCl, 900mM Particle containing SwIVA, 490mM NaCl eluent SDS-PAGE purity assays centrifuge with sucrose density gradient in NaCl eluents Or the swine influenza virus of heparin affinity chromatography post purifying approaches.Amount to the swine influenza virus liquid of 1000 milliliters of harvest.

3.2.4 it is concentrated by ultrafiltration, dialysis

From ion exchange column elution virus liquid with cutoff value be 700KD films be concentrated by ultrafiltration 100 times, use simultaneously 0.02M Tris-HCl (pH 7.5) dialyse.

3.2.5 molecular exclusion chromatography

The chromatographic column column of 10ml concentration swine influenza virus stoste loadings FF-Sepharose 6 (are purchased from GE Healthcare companies).The equilibrium liquid of chromatographic column is 0.02M Tris-HCl (pH 7.5).Flowed with equilibrium liquid with 0.8ml/min Speed elution, while eluent is detected in OD₂₈₀Absorption value, collect elution first peak.First peak is the swine influenza virus of purifying.

3.2.6 it is concentrated by ultrafiltration, dialysis

Viral purification liquid is used into film of the cutoff value for 700KDa, using phosphate buffer (50mM phosphate buffers, 150mM NaCl, pH 7.5) 2 times of dialysis are concentrated by ultrafiltration, the swine flu totivirus liquid of 100 milliliters of inactivation purifying is obtained, for weight The preparation of structure virion.

4th, the preparation of swine influenza virus reconstructed volume and the Non-covalent binding with PRRSV antigens

The preparation of 4.1 phospholipid dispersions

It is mixed with the 0.01M Tris/HCl (pH 7.3) containing 0.1M NaCl in homogenizer containing phosphatide and courage The dispersion liquid of sterol mixture, wherein containing 75% in percentage by weight in described phosphatide and the mixture of cholesterol Phosphatidyl choline (lecithin, purchased from SIGMA companies of the U.S.), 20% phosphatidyl-ethanolamine (cephalin, purchased from U.S. SIGMA Company) and 5% cholesterol (being purchased from SIGMA companies of the U.S.) dispersion liquid, wherein all phosphatide account for 1-2% (w/v).By cholic acid Sodium is (from acetone/water (4：1, V/V) recrystallization obtains) added as acid in phospholipid dispersions, final concentration is at least 1.3% (w/v) To disassemble the sandwich construction of not ultrasonic phospholipid dispersions.

The preparation of 4.2 swine influenza virus reconstructed volumes

700ml glycol list dodecyl ethers containing 0.1M eight are added into the swine influenza virus H1N2 strain virus liquid of purifying (octaethyleneglycol mono (n-dodecyl) ether) (C.sub.12E.sub.8) (is purchased from Japanese Nikko Chemicals companies), 7.9mg/ml NaCl, 4.4mg/ml sodium citrate, 2.1mg/ml MES, 1.2mg/ml N- ethoxys- Piperazine-N'-2- ethyl sulfonic acids (N-hydaxylethyl-piperazine-N'-2-ethane sulfonic acid) aqueous solution (pH 7.3).Mixture centrifuges 30min on ultracentrifuge with 170000g, collects supernatant, obtains viral envelope proteins, i.e., Hemagglutinin (HA) and neuraminidase (NA).The supernatant of acquisition is added to the phospholipid dispersions of above-mentioned acquisition, it is outstanding in 4 DEG C of stirrings Supernatant liquid at least 1 hour, loading Sephadex G-50 chromatographic columns (80 × 15 cm).Column equilibration liquid and post eluent save with 2.2.3 It is identical, flow velocity 320ml/h.Pillar is placed to be connected (frequency 50kHz+-10%) with Ultrasound Instrument in a water bath, ultrasound shake per minute Swing and produce within 10 seconds small individual layer influenza virus reconstructed volume.Reconstituted influenza virion and cholesterol micro-capsule flow out in outer water section.Receive Collect void volume part, the merging of the reconstructed volume containing swine influenza virus chromatographs again under similarity condition, obtain swine influenza virus Reconstructed volume, average diameter 150nm.First time Sephadex G-50 is chromatographed, and 1% cholesterol is stranded on post, and phosphatide/courage is solid Alcohol mol ratio>50.Dialysed 12 hours at 4 DEG C after second Sephadex G-50 chromatography, cholesterol amount reduce to detectable limit with Under, phosphatide/cholesterol mol ratio>500.

The preparation of 4.3 porcine reproductive and respiratory syndrome viruses-swine influenza virus reconstituted virosomes

The PRRSV suspensions and swine influenza virus reconstructed volume that the inactivation of above-mentioned acquisition is purified mix, gently shake resuspension, 20 DEG C are gently mixed the surface for making PRRSV be adsorbed in swine influenza virus reconstruct virion by Van der Waals force for 48 hours.Obtain pig Reproductive and respiratory syndrome virus-swine influenza virus reconstituted virosomes.

5 covalent bonds prepare porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct Virosome particles

5.1 Thiolation PRRSV preparation

5.1.1 the preparation for purifying the PRRSV totivirus antigens of inactivation is carried out according to the method described above.

5.1.2 PRRSV's is Thiolation

5ml inactivation purifying PRRSV, which is dissolved in 0.1M phosphate buffers (pH 7.5), makes its concentration be 5mg/ml. N- Succinimidylpyridyl dithiopropionate (SPDP, purchased from power ＆ light company of the U.S.) and ethanol mixing make the concentration be 6mg/ml, 150 μ l gentle agitations are taken, be slowly added to the phosphoric acid solution that 5ml contains PRRSV with syringe, make SPDP's and PRRSV Mol ratio 15:1, while keep concentration of alcohol to prevent albuminous degeneration below 5%.(20 DEG C) of mixture room temperature is reacted 30 minutes. After reaction terminates, Sephadex G- are balanced with 0.05M sodium citrates, 0.05M sodium phosphates, 0.05M sodium chloride (pH7.0) respectively 50 pillar is purified three times.

The preparation of 5.2 swine influenza virus reconstructed volumes

The preparation of swine influenza virus reconstructed volume is carried out according to 4.1 and 4.2 method, and difference is：Using phosphatidyl Before choline, phosphatidyl choline (PE) is crosslinked with SPDP：15mg PE (20 μm of ol), which are put into 5ml vials and dried, (to be passed through Molecular sieve drying).Dried PE is redissolved in 2ml chloroform, then adds and contains 30 μm of ol triethylamine (TEA) 1 milliliter of absolute ethyl alcohol of (3mg), 30 μm of ol SPDP (10mg).It is straight that 1-2 hours are stirred at room temperature under logical condition of nitrogen gas in mixture Completed (without single lecithin) to reaction.Reaction product is dried on a rotary evaporator.Product is resuspended in chloroform, loading silicic acid Chromatographic column.

Silicic acid chromatography column preparation method：2g silicic acid is dissolved in 10ml chloroforms, loads 10ml and fills up fiberglass plastic syringe, Dry it and mouth disposes the disposable three-way stopcock of plastics on the injector.

Silicic acid chromatography method：After loading, pillar mixes first with 4 milliliters of chloroforms, then with 4 milliliters of serial chloroform-methanols Thing elutes, for the first time using chloroform：Methanol ratio 4:0.25 (v/v) mixture, use chloroform for the second time：Methanol ratio 4:0.75 (v/v) mixture, finally using chloroform：Methanol ratio 4:1 (v/v) mixture.Collect eluent, merging, at reduced pressure conditions Concentrated with Rotary Evaporators.Obtain PE-SPDP.

Detection method：2ml milliliter eluents are collected, with silicic acid glue thin-layer chromatography and chloroform-methanol-water (volume ratio 65: 25:4) detection purified runs a good foot than lecithin, and spot use can use molybdophosphate or iodine staining visual.

5.3 swine influenza virus bodies and the coupling of PRRSV antigens

5.3.1 the reduction of Thiolation PRRSV totivirus antigen

Thiolation PRRSV totivirus antigen restoring method：By Thiolation PRRSV in 0.2M citrate phosphate buffers The pH of totivirus antigen (PRRSV-SPDP) is adjusted to 5.5 with 1M HCl, and 10 microlitres of 2.5M are added by every milliliter of viral antigen solution Dithiothreitol (DTT) solution (2.5M DTT).Solution is placed 30 minutes, and Sephadex G-50 are balanced with PBS (pH7.0) buffer solution Separate DTT.To prevent disulfide bond from aoxidizing, all buffer solutions are steeped with nitrogen system removes oxygen, and albumen is collected in having nitrogen environment.

5.3.2 the preparation of porcine reproductive and respiratory syndrome virus-swine influenza virus reconstituted virosomes

Thiolation PRRSV totivirus antigen after the reconstruct swine influenza virus body of above-mentioned acquisition and reduction is stirred at room temperature Mix overnight.Obtain porcine reproductive and respiratory syndrome virus-swine influenza virus reconstituted virosomes.

5.4 swine influenza virus bodies and the coupling of PRRSV antigens

Section 4.3 as described above, but do following adjustment of the preparation of PRRSV- swine influenza virus bodies：PRRSV antigens not with SPDP With reference to PRRSV surfaces GP5, GP4, GP3, GP2 have had disulfide bond and reacted as follows as free disulfide bond:Use 0.1M 5ml PRRSV solution, the mg/ml of final concentration 5 is made in phosphate buffer (pH 7.4).It is micro- that every milliliter of PRRSV protein solution adds 10 2.5M DTT solution is risen, room temperature places 30min, PBS (pH 7.0) balance Sephadex G-50 column separating purifications. 1mg inactivation purifying PRRSV suspensions, are added in swine influenza virus body (0.002M influenza viruses membrane phospholipid), obtain pig breeding with exhaling Inhale syndrome virus-swine influenza virus reconstruct virion.

The preparation of 6 porcine reproductive and respiratory syndrome virus-swine flu reconstruct virosomes vaccine

Porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virion is diluted to containing 0.2- with PBS (pH 7.4) The vaccinogen liquid of 0.5 microgram/ml PRRSV antigens, through 0.2 μm of film (Millipore) aseptic filtration, with Heat-resistant stable adjuvant Compound is mixed and made into vaccine semi-finished product.

The stable adjuvant compound of embodiment 2, porcine reproductive and respiratory syndrome totivirus-swine flu reconstruct virosomes vaccine Preparation and quality control method

1st, the preparation and calibrating of BCG vaccine lysate

Strain BCG vaccine (BCG) D2BP302S11, provided by Chinese medicine and biological product determination research institute, made by biology Product production calibrating establishes seed lot with Bacterial Strains Managing code.Working seed lots to microorganism collection was not to be exceeded for 12 generations, in Su Tongpei Support base on BCG vaccine should float on surface, for wrinkle more, be yellow mycoderm.It is virulence test, negative without poisonous BCG vaccine result of the test. Freeze-drying lactobacillus preserves below 2-8 DEG C of preservation, -70 DEG C of liquid spawn.

BCG vaccine D2BP302S11 logical 37 DEG C of synthetic medium static gas wave refrigerators 2-3 weeks of improvement Soviet Union, culture is at 121 DEG C Carry out sterilization processing within 30 minutes, harvest thalline with preparative low speed centrifuge, PBS (pH 7.4) is washed 2 times, is suspended by 2 g/ml In EDTA containing 8mM, protease inhibitors, DNA enzymatic, RNase PBS in, crush bacterium with bead, during which rapid dyeing see Broken situation is examined, until 90% bacterial cell disruption, the unbroken cell of 3000g centrifugations, insoluble cell wall constituent, harvest is split Solve supernatant and obtain whole cell lysate (WCL), supernatant adds benzene through 0.2 μM, low protein bound membrane filtration by 3.0g/L Phenol.Containing water-soluble albumen, lipid and carbohydrate in BCG lysate WCL stostes, level of endotoxin should be not higher than 0.002 μ g/mg albumen, in 2-8 DEG C of preservation.BCG lysate WCL stostes can preserve 5 years in 2-8 DEG C of preservation.

2nd, the preparation of poly-D-lysine hydroxymethyl cellulose compound

Compound polyICLC is synthesized by this experiment, poly-D-lysine Poly-L-Lysine (molecular weight 1000- 4000Da, Cat.No.P0879), poly IC (Cat.No.P0913) and hydroxymethyl cellulose (carboxymethylcellulose, low viscosity, Cat.No.C5678) is purchased from Sigma-Aldrich companies (St. Louis,Mo).Poly IC (mean molecule quantity 200000-500000Da, 500mL；4.0mg/mL), poly-D-lysine (poly- L-lysine, 250ml；6.0mg/mL) and 2% hydroxymethyl cellulose (carboxymethylcellulose, 250mL) is by nothing It is prepared by thermal source 0.85%NaCl.Poly-D-lysine hydroxymethyl cellulose compound (Poly ICLC) preparation method is：poly IC Anneal within 1 hour in 71 DEG C of heating and slowly cool down again, add poly-D-lysine containing 6.0mg/mL and 2% hydroxymethyl cellulose etc. The physiological saline of volume, poly-D-lysine hydroxymethyl cellulose compound is obtained, i.e., stable adjuvant compound, adjust its mixed liquor In ultimate density be 1mg/mL, 4 DEG C are standby.

3rd, the preparation of stable adjuvant compound

Calculate to be 20mg/100kg, vaccine effective dose 5mL by clinical test.

Specific method：Into 100ml 50mM phosphate buffers, add 0.6g arginine and glutamic acid etc. part by weight Mixture, 2.2g poly-D-lysine hydroxymethyl celluloses compound (Poly ICLC), 0.03mg BCG vaccines lysate, 5g malt Sugar, 6.5g D-sorbites, 0.4g urea, 0.02g EDTA, 0.005g Tween-20s, pH value 8.0 is adjusted, stable assistant is prepared Agent compound.

4. porcine reproductive and respiratory syndrome totivirus-swine influenza virus reconstructed volume vaccine formulation and quality control method

4.1 vaccine formulation

By the inactivation obtained according to the method for embodiment 1 purifying porcine reproductive and respiratory syndrome totivirus-inactivation Purification of Pig stream Influenza Virus reconstructed volume, which is received, to be added in preparation-obtained stable adjuvant compound, is made vaccine semi-finished product, 5 milliliters every dose：Containing pig Reproductive and respiratory syndrome totivirus-μ the g of swine influenza virus reconstructed volume antigen 30.

4.2 quality control method：

4.2.1 inactivation purifying PRRSV totivirus detection method of content

4.2.1.1 prepared by the porcine reproductive and respiratory syndrome virus polyclonal antibody of affinitive layer purification

Inactivation purifying whole virus particles are super to exempt from rabbit, obtains the antibody serum of high titre, serum is through saturated ammonium sulfate, G eggs White analysis, the Sephorose-4B column chromatographies purifying of porcine reproductive and respiratory syndrome purified virus coupling, it is pure to obtain affinity chromatography The antibody of change, indicate that the antibody and porcine reproductive and respiratory syndrome virus reaction are special, sensitive through examining.

Inactivation purifying porcine reproductive and respiratory syndrome virus whole virus particles antigenic content measure：With affinitive layer purification Indirect ELISA standard measure porcine reproductive and respiratory syndrome disease is established based on porcine reproductive and respiratory syndrome virus polyclonal antibody The content of poison.Utilize the level of parallel method measure pig body neutralizing antibody and the relation of purifying totivirus amount of antigen, the pig of purifying Reproductive and respiratory syndrome virus can produce protectiveness neutralizing antibody for 20 μ g, it is determined as 20 units, can be estimated by indirect elisa method Calculate the potency of inactivation porcine reproductive and respiratory syndrome vaccine.

4.2.1.2 total protein detects

The operating guidance that total protein concentration concentration is provided using Bradford protein assay kits by manufacturer is carried out, ox Seralbumin is as standard items.

4.2.1.3 PRRSV granule numbers detect：PRRSV samples are respectively walked using technique and carry out real-time fluorescence system detectio PCR (Bio-Rad iQ5Real Time PCR Detection System),20μL(iQ SYBR Green Supermix； Bio- Rad) reaction volume includes 10 μ L mixtures, 1 μ L Swine serum sample cDNA extracts, and 8.5 μ L are free of the water of RNA enzymes, using drawing Thing porcine reproductive and respiratory syndrome virus O RF5 the primer ,-TCTTGCTTCTGGTGGCTTTT-3 ' of forward primer 5 '；Reversely draw Thing 5 '-CATGTTTGATGGTGACGAGG-3 ', each 2.5 μ L, PRRSV1 is set up per secondary response：The standard curve of 10 dilutions.Instead Answer condition：94 DEG C of 5min, 40 circulations, every time circulation, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 45 DEG C of extension 45s.It is anti-every time The PRRSV contents that should be assessed change into cycle threshold Ct, true by Bio-Rad iQ5 qPCR softwares and calibration curve coefficient correlation Determine viral level (copies/ml).

4.2.1.4 PRRSV structural proteins detection methods：

SDS-PAGE electrophoresis is used to detect PRRSV purification effects, often walks 20 microlitres of refined solution sample and adds on 5 × 5 microlitres Sample buffer solution, 10 minutes albuminates, the 10-20 microlitres of polyacrylamide gel of each sample loading 15%, electricity are heated at 100 DEG C Coomassie liquid (0.04%Serva indigo plants, 25% isopropanol, 10% acetic acid) dyeing 10min is used in swimming after terminating, with 10% acetic acid It is high-visible to be washed till protein band.

Electronic Speculum detects：50 microlitres of purifying PPRSV are positioned over static 1min in copper mesh.Residual liquid is removed with filter paper, is used 3%phosphotungstic acid dye 1min.Drying at room temperature, project electric Microscopic observation.

4.2.1.5 PRRSV quantitative analyses：

Each step virus liquid dilutes (10 with without DMEM10 times of cow's serum^-0-10^-5), each sample MARC-145 cells paving Full titrates in triplicate on 96 orifice plates.Each dilution factor sample adds four holes, per 100 microlitres of hole.96 orifice plates 37 DEG C, 5%CO₂Incubator is incubated 1h, outwells liquid, 200 microlitres of cell maintenance mediums is added per hole, in 37 DEG C, 5%CO₂Incubator is incubated 4 days directly To there is cytopathic effect.logTCID₅₀/ mL is calculated by Reed-Muench formula.

4.2.2 porcine reproductive and respiratory syndrome totivirus-swine influenza virus reconstructed volume particle (PRRSKPWV-SwIVA- RV) size, form and nano particle Efficiency testing

Determined with ESEM (Electronic Speculum Hitachi S-3500N), observation nano particle size, form.To be measured freeze is received Rice grain is placed on the coated adhesion tablet of golden platinum using ion coating instrument, and 10KV is observed in Electronic Speculum.Nanometer embedding protein efficiency： Lyophilized PRRSKPWV-SwIVA-RV 10mg add 0.1N NaOH, and 37 DEG C are handled 1 hour, the harvest that is vortexed supernatant, use 0.1N NaOH prepares BSA and BCA protein detection kits (being purchased from Pierce companies of the U.S.) detection protein concentration.

PRRSKPWV-SwIVA-RV feature detections：Scanning electron microscopic observation PRRSKPWV-SwIVA-RV is smooth spherical shape, Size is between 200-300nm.It is defined by PRRSKPWV albumen usage amounts, the yield of PRRSKPWV-SwIVA-RV nano particles For 57.3 ± 2.1%, the mV of nano particle NP surface electrostatics lotus -34.It is defined by SwIVA protein contents, average protein in nano particle Content 0.50-0.55% (w/w), represent virion formation efficiency 50-55%.

PRRSKPWV-SwIVA-RV nanoparticle features under focusing microscope：From the SPF pigs of 3 4-6 week old, health Macrophage (lavation is separated and collected in BAL fluid (Bronchoalveolar lavage fluid, irrigating solution) Liquid-MNC), it is inoculated with 24 orifice plates, inoculum density 1 × 10⁶Cell/mL.24 orifice plates fill containing the cover glass for being coated with more polylysines 37 DEG C of 5%CO of washing lotion-MNC plates₂It is incubated 1 hour, removes the cell liquid being not bound with, cover glass is gently washed with PBS.Will be lyophilized PRRSKPWV-SwIVA-RV be resuspended in the DMEM containing 10%FBS, its concentration is reached 0.2 microgram/mL.Addition contains lavation Liquid-MNC culture plate, 37 DEG C are incubated 3 hours.It is uninfected by PRRSV or with 0.1MOI infection PRRSV (HP-PRRSV-JXM-F5) Irrigating solution-MNC plates are in 37 DEG C of 5%CO₂Incubator is incubated 12 hours, 15 minutes is fixed on ice with 3% paraformaldehyde, with 0.1% Triton X-100 are impregnated with 15 minutes, are closed 1 hour with containing 5%BSA, 0.2%triton X-100PBS at room temperature.Then it is thin Born of the same parents add anti-PRRSV nucleocapsids monoclonal antibody specific mAb SDOW17 (U.S. rural area Technology Co., Ltd. product) and endogenous The anti-pig human antibody of cross reaction (is dissolved in the PBS containing 1%BSA, 0.1%triton X-100, purchased from U.S. Santa-Cruz public affairs Department), room temperature is placed 1 hour, adds sheep anti-mouse igg Alexa Flour488 and the anti-goat-anti body Alexa flour 633 of donkey (purchases From Invitrogen companies of the U.S.), it is incubated at room temperature 1 hour.Cell washs and with containing 2.5% between antibody procedures are added DABCO (being purchased from Sigma Co., USA) liquid handling.The cover glass of dyeing moves on to slide and seen under Leica focusing microscopes Examine, image focuses on software analysis using Leica.With 96 orifice plates of U-shaped bottom, 1 × 10 is contained per hole⁶Cell.Irrigating solution-MNC plates Above-mentioned similar process can be used, obtains similar comparative results.

Flow cytometer determines that CD80/86 cells in vitro absorbs inactivation purifying PRRSV totivirus-swine influenza virus reconstruct disease Malicious body (PRRSKPWV-SwIVA-RV) nano particle：Antigen presenting cell CD80/86 is inoculated with 24 orifice plates, inoculum density 1 × 10⁶ Cell/mL.Inactivation purifying PRRSV totivirus (PRRSKPWV) is added into CD80/86 respectively or inactivation purifying PRRS is entirely sick Poison-swine influenza virus reconstruct virion (PRRSKPWV-SwIVA-RV), make PRRSV protein contents respectively reach 2 micrograms/mL, 0.2 microgram/mL, 0.02 microgram/mL.It is incubated 3 hours in 37 DEG C of 5%CO2 incubators.With 0.1MOI infection PRRSV (HP- PRRSV-JXM-F5), in 37 DEG C, 5%CO₂Incubator is incubated 12 hours.Using be uninfected by PRRSV CD80/86 plates cell as pair According to.Combined after washing with biotin combination human CTLA 4 rat immune globulin fusion protein (being purchased from Ancell companies of the U.S.), PE- CD172 (being purchased from Southern Biotech companies of the U.S.) and Streptavidin PerCP-Cy5.5 are dyed successively, with 1% Paraformaldehyde is fixed, and is analyzed using FACS Aria II flow cytometers (being purchased from BD Biosciences companies).

Above-mentioned in vitro test is shown under normal physiological conditions, inactivation purifying PRRSV totivirus-swine influenza virus reconstruct disease Malicious body (PRRSKPWV-SwIVA-RV) intermittently discharges PRRSV antigens within several weeks, can be by porcine alveolar macrophage and antigen Presenting cell CD80/86 effectively absorbs.

4.2.3 the measure of the protein content in BCG vaccine whole cell protolysate (WCL)

Using Kjeldahl's method (Chinese Pharmacopoeia three, version annex VI B in 2005)；Level of endotoxin detects in accordance with the law in WCL (Chinese Pharmacopoeia three, version annex XII E in 2005)；Determination of polysaccharide, without poisonous BCG vaccine experiment, discrimination test, sensitization Effect test is carried out by Chinese Pharmacopoeia three (version in 2005) p254-p255 method；Nucleic acid content measure uses ultraviolet-visible AAS (Chinese Pharmacopoeia three (version in 2005) annex II A；Sterility testing is attached by Chinese Pharmacopoeia three (version in 2005) The method for recording XII A；Undue toxicity detection is checked in accordance with the law by three (version in 2005) annex XII F of Chinese Pharmacopoeia.

The porcine reproductive and respiratory syndrome intact virus of embodiment 3-swine influenza virus reconstruct virosomes vaccine stability and effect Phase

3.1 vaccine semi-finished product liquid freezing state stability inferiors

Porcine reproductive and respiratory syndrome intact virus-swine influenza virus reconstructs virosomes vaccine stoste according to the side of embodiment 1 Prepared by method, stable adjuvant compound and vaccine antigen preparation method are prepared according to method in embodiment 2.

Vaccine semi-finished product, which are stored in polypropylene vial, is placed on -35 DEG C, -70 DEG C, and inactivation Purification of Pig breeding is detected every March Virosome particles are reconstructed with breathing syndrome intact virus-swine influenza virus, as a result such as table 1 below.

The vaccine semi-finished product of table 1 are stored in freezing state stability inferior

The result of table 1 shows that porcine reproductive and respiratory syndrome intact virus-swine influenza virus reconstructs virosomes vaccine semi-finished product After -35 DEG C, -70 DEG C store December, no signs of degradation is analyzed through laser power grating (DLS), and granular size is mainly distributed on 200-300nm scopes, without degradation fragment, keep porcine reproductive and respiratory syndrome intact virus-swine influenza virus reconstruct virus Body nano particle is complete, also virus-free coacervation.Illustrate porcine reproductive and respiratory syndrome inactivation purified nanotubes particle vaccines half Finished product under freezing conditions, can store 1 year stabilization, also illustrate the stable adjuvant compound of the present invention in vaccine preparation process to epidemic disease The protective effect of seedling Effective Antigens.

3.2 vaccine semi-finished product are in 5-25 DEG C of stability

The liquid of some batches of stable adjuvant porcine reproductive and respiratory syndrome vaccine semi-finished product is stored in 5 DEG C, 25 DEG C, every 30 days detection porcine reproductive and respiratory syndrome intact viruses-swine influenza virus reconstruct Virosome particles protein content, it is as a result as follows Shown in table 2：

23 batches of semi-finished product different temperatures storage porcine reproductive and respiratory syndrome intact viruses of table-swine influenza virus reconstruct disease Malicious body granule protein content

Above-mentioned result of the test shows that stablizing the addition of adjuvant compound makes porcine reproductive and respiratory syndrome intact virus-swine flu Viral reconstruct virion keeps particle complete at 4-8 DEG C, at least March, and vaccine is stored at least March at 5 DEG C, and 25 DEG C are stored 30 days surely It is fixed.Also the stable adjuvant compound of the explanation present invention is complete to inactivation purifying porcine reproductive and respiratory syndrome in vaccine storage, cold chain Whole virus-swine influenza virus reconstructs the protective effect of this vaccine Effective Antigens of Virosome particles.

The stability of 3.3 porcine reproductive and respiratory syndrome intact viruses-swine influenza virus reconstruct virosomes vaccine finished product

Neutralizing antibody detection method：Antiserum doubling dilution, respectively with 2 × 10³TCID50/mL virus liquid mixing, 37 DEG C The orifice plate of individual layer MARC-145 cells 96 is covered with hatching 1 hour, immigration, cytopathic effect after analyzing 7 days, prevents 50% cell The inverse of the serum dilution of lesion effect is then neutralizing antibody titers, as a result as shown in table 3.In vaccine-induced sow protectiveness It is effective with antibody titer 32-68.

33 batches of vaccines of table are stored in effect (2-128/mL) and effect (U/mL) under different temperatures

Result is shown in table 3, and porcine reproductive and respiratory syndrome intact virus-swine influenza virus reconstructs virosomes vaccine 5 DEG C preserve the effect phase be 2 years, at normal temperatures store March do not fail, 37 DEG C of effect phase is January.The good stability of vaccine is shown, Also the good stability of the stable adjuvant compound of the explanation present invention.

Therefore, vaccinogen liquid, the semi-finished product of the stable adjuvant compound of addition and finished product are stored all keep at different temperatures It is stable, show by phosphate buffer, must and non-essential amino acid blend, maltose, D-sorbite, EDTA, urea, tell Warm -20 poly-D-lysine hydroxymethyl celluloses, the stable adjuvant compound of BCG vaccine lysate composition can be protected effectively in pig Reproductive and respiratory syndrome intact virus-swine influenza virus reconstruct Virosome particles antigen is complete under liquid, freezing state Property, porcine reproductive and respiratory syndrome intact virus-swine influenza virus reconstruct virion of stability and invention, semi-finished product, into The stability of product.Vaccine imitated the phase up to 2 years, under thermal extremes, imitated 1 month phase.Semi-finished product, finished product are in normal temperature and 4-8 DEG C Keep stable in the long period, be convenient for large-scale preparation and transport.

The porcine reproductive and respiratory syndrome intact virus of embodiment 4-swine influenza virus reconstructs virosomes vaccine to piglet, mother The security of pig, immune programme for children, immune effect

Method：

4.1 porcine reproductive and respiratory syndrome Virus cultures

Attack poison uses strain JX0708-F12, by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences Jing Zhizhong researcher point From, provide.Malicious reproductive and respiratory syndrome virus Qinghai strain QH-08 is attacked in infection, and (gene accession number KU201579.1 passes in Marc145 cells 50 generations of generation) Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science Zhang Jie researcher separation.Attack poison MARC-145 cells, flask culture, Plaque clone purification three times.Culture medium is that the DMEM condition of culture containing 10% hyclone is 37 DEG C, cultivates 4 under the conditions of 5%CO2 My god.Virus titer is determined with primary pulmonary alveolar macrophage cultivation, is diluted when gilt attacks poison with the MEM containing 4% calf serum To 3.0logTCID50/ml, nasal cavity instillation 2ml.Sow is diluted to 5.0TCID50/ml, nasal cavity instillation 2ml when attacking poison.

4.2 are grouped, are immunized, attacking poison, observation

The porcine reproductive and respiratory syndrome intact virus of the present invention-swine influenza virus reconstruct virosomes vaccine (prepared by PRRSKPWV-SwIVA-RV, the method for the embodiment 2), vaccinogen liquid (PRRSKPWV- for being not added with stablizing adjuvant compound It is prepared by SwIVA-RV, embodiment 1), commercial inactivated vaccine, nasal membrane instil inoculation.Vaccine (the HP- of nasal membrane inoculation PRRSV-JXM-F5) used time is diluted to 5 milliliters with water for injection.

6 week old gilt of screening PRRSV negative antibodies 450 is detected with kit, is divided into 8 groups, 1-3 groups intranasal vaccination hair Bright porcine reproductive and respiratory syndrome intact virus-(PRRSKPWV-SwIVA-RV+ is steady for swine influenza virus reconstruct virosomes vaccine Determine adjuvant compound), 1 group of dosage is 1 dose, and 2 groups of dosage are 2 doses, is spaced 2 weeks, 3 groups of dosage are 3 doses, 4 groups of intranasal vaccination vaccines Stoste (PRRSKPWV-SwIVA-RV), 3 doses, it is spaced 2 weeks.5 groups of intranasal instillation inoculation water, instil 3 times, every time 5 milli Rise.6 groups of intranasal instillation PRRSV commercialization inactivated vaccines, the vaccine (PRRSKPWV-SwIVA- of the intranasal inoculation invention of instiling of 7-9 groups The stable adjuvant compounds of RV+), 7 groups of 1 dose of immunizing doses, 8 groups of dosage are 2 doses, are spaced 2 weeks, 9 groups of dosage are 3 doses, are spaced 2 weeks. Each group the difference of infection attack poison and time because dividing subgroup.Experimental design and packet are shown in Table 4.

The gilt experimental design of table 4

30 after the completion of last 1 dose of gilt, attack poison within 60,90,120,150 days.The health status of immune fore-and-aft observing pig, body Change again, Temperature changing, taking blood within 2 weeks with after the completion of finally one be immunized before being immunized, detecting the neutralizing antibody titers of serum, sight Examine the health status for attacking pig after poison infects.Carry out attacking malicious infection respectively within 30,60,90,120,150,180 days after each group is immune, The strong poison of challenge infection JX0708-F12, QH-08 are malicious by force respectively.Whether observation attacks the health status of malicious pig after infecting, viremia virusemia Occur and time of origin, detection attack poison after 2 weeks the neutralizing antibody of serum, observation pig attack body temperature and clinical condition after poison in 2 weeks Shape, dead and Survival.

From the different weight age at least in the sow at 6 monthly ages, the pig 80 for screening PRRSV negative antibodies is detected with kit Head is divided into three groups.One group of one dosage inactivation purified nanotubes particle vaccines (HP-PRRSV-JXM- of nasal cavity instillation in 1 week before childbirth F5 strains), it is spaced and carries out within 2 weeks 2nd time, the 3rd nasal cavity drop, 1 vaccinating agent amount of antigen and stablizes adjuvant amount as 10 times of gilt, body Product is 5 milliliters.Two groups of ultrasound detections confirm farrowing sow, in the epidemic diseases of intranasal 3 doses of inventions of instiling in 30-45 days of period of pregnancy Seedling, three groups with water for injection respectively in 0,14 day, intranasal instillation in 48 days.

The health status, changes of weight, Temperature changing of fore-and-aft observing pig is immunized in sow, before immune and last one immune After the completion of take blood within 2 weeks, detect the neutralizing antibody titers of serum, observe it is immune after pig body side reaction.It is highly pathogenic to attack poison China Porcine reproductive and respiratory syndrome virus QH-08 is malicious by force, observation attack poison infection after pig health status, viremia virusemia whether occur and Time of origin, sow is immunized in 60 days, 90 days, infection attack in 120 days.By the life for the health Evaluation sow for observing piglet Grow behavior.The effect of the survival number measure vaccine of piglet is produced after 28 days by relative immunity sow and rather Farrowing.

Infection attacks malicious restrovirus detection and uses RT-PCR methods, and total serum IgE is extracted by commercial kits, numerous using primer pig Grow the primer with breathing syndrome virus ORF5：

- the TCTTGCTTCTGGTGGCTTTT-3 ' of forward primer 5 '

- the CATGTTTGATGGTGACGAGG-3 ' of reverse primer 5 '

The first round expands the sensitivity 10 of the method²TCID50, expand 499bp DNA fragmentation, using the product of the first round as Template carry out second wheel amplification, every time 35 times circulation, every time circulation, 94 DEG C denaturation 45s, 55 DEG C annealing 45s, 72 DEG C extension 45, Detectable 1-10 copies/ml serum-virus.

PRRSV specific antibody ELISA methods：From pig body collect separation serum be stored in -20 DEG C it is standby, with commercial ELISA Kit detection antibody (PRRS 2XR, IDEXX), by specification operates at room temperature, is determined at 650nm Absorbance value (microplate readers of Bio-Rad 680), determine whether PRRSV antibody is positive by positive and negative sample ratio Property.Positive OD/ feminine genders OD is more than 2.1 for positive value.

Serum neutralizing antibody detection method：Serum doubling dilution, respectively with 2 × 10³TCID50/mL virus liquid mixing, 37 DEG C hatching 1 hour, the orifice plate of individual layer MARC-145 cells 96 is covered with immigration, cytopathic effect after analysis 7 days, prevents 50% cell The inverse of the serum dilution of lesion effect is then neutralizing antibody titers.

As a result：

All 3 doses of collunariums, 2 doses, the piglet that 1 dose of immune piglet body temperature is normal, average weight gain is more than water for injection instillation, System and local side reaction is not observed.Illustrate that invention vaccine does not influence growing for piglet, nasal cavity instillation vaccine pair Gilt safety.

All 3 doses, 2 doses it is intranasal instil immune sow and 1 dose of intranasal immune farrowing sow mean body temperature that instils it is normal, flat Weightening is more than the sow of water for injection, and heating and nasal cavity symptom is not observed in Nasal immunization sow, also without other side reactions.

3 doses of intranasal immune 10 sows being pregnant of instillation, it is average to be farrowed 9.8 per nest, after giving a birth 28 days, piglet body Temperature, weightening are normal, no stillborn foetus and piglet hypoplasia phenomenon, and the intranasal instillation of 3 doses of vaccine of invention is immune to be used, to farrowing sow Gestation and farrowing do not influence, on Pig embryos also without influence.

Above result of the test shows invention vaccine, and intranasal inoculation of instiling is safe to gilt, sow, farrowing sow three times.

1 group of piglet is before poison is attacked, and 5/10 generates PRRSV specific antibodies, and 2/10 gilt produces neutralizing antibody, exempted from 30 days afterwards, JX0708-F12 attacked poison, there is 10/10 generation viremia virusemia, and the average virus mass formed by blood stasis time is 17 days, the production of 4/10 gilt Raw neutralizing antibody, the gilt of average titer 32,1/10 are dead；Poison is attacked after immune within 60 days, there is 10/10 generation viremia virusemia, is put down The equal viremia virusemia time is 18 days, and 2/10 gilt produces neutralizing antibody, and the gilt of average titer 16,2/10 is dead；1 group In, 1 dose exempt from after attack poison infection gilt with JX0708-F12 within 90 days, have a 10/10 generation viremia virusemia, during average virus mass formed by blood stasis Between be 21 days, 1/10 gilt produces neutralizing antibody, and the gilt of titre 16,3/10 is dead；7 groups of gilt, 1 dose of nasal cavity instils Carry out QH-08 and attack poison within 30 days after immune.There is 6/10 gilt to generate porcine reproductive and respiratory syndrome disease after vaccine instillation is immune Malicious antibody, 1/10 gilt generate QH-08 neutralizing antibodies, antibody titer average out to 8.After QH-08 attacks poison, 2/10 it is small Sow can detect neutralizing antibody, average titer 16, maintain by 60 days.10/10 gilt generates viremia virusemia, average hair The raw time is 18 days, 4 gilt death；Poison is attacked with QH-08 infection within 60 days after immune, it is anti-that 2/10 gilt generates neutralization Body, titre 32.10/10 gilt generates viremia virusemia, and time of origin is 21 days, 2 gilt death；90 after immune It attacks poison, and 10/10 gilt has viremia virusemia, and time of origin is 22 days, 3 sow death, and 3/10 gilt is dead.

There are 9/10 generation PRRSV specific antibodies in 2 doses of intranasal immune gilt of instiling, the gilt for having 8/10 produces JX0708-F12 neutralizing antibody, antibody average titer 16,90 days JX0708-F12 challenge infections after immune, whole gilt There is viremia virusemia, time average out to 15 days, 7/10 gilt produces high titre JX0708-F12 neutralizing antibodies, average to neutralize The gilt of antibody titer 32,10/10 all survives；JX0708-F12 infection in 120 days after exempting from, viremia virusemia average time are 16 My god, 6/10 gilt neutralizing antibody level remains at 16, still positive for neutralizing antibody.

2 doses of intranasal immune, QH-08 that instil are attacked in the gilt of poison, are completed 2 doses of immune gilt of instiling and are all generated spy Different in nature PRRSV antibody, there is 3/10 piggy to generate to QH-08 neutralizing antibodies, average titer 16.Carry out QH-08 and attack within 60 days after exempting from The gilt of poison, wherein 9/10 gilt has viremia virusemia, average time is 15 days, and 5/10 gilt is dead, 5/ 10 gilt produces neutralizing antibody, average titer 16.120 days postoperative infection QH-08,6/10 small mother is immunized in 2 doses of intranasal instil Pig neutralizing antibody level remains at 32, viremia virusemia time of origin average out to 17 days, and 2/10 gilt is dead.150 after immune After poison is attacked in its QH-08 infection, 5/10 gilt produces antibody.3/10 gilt is dead.

Serum neutralizing antibody titers after the immune gilt of water for injection is immune are detected as less than 8, PRRSV specific antibodies Feminine gender, synchronous and other immune 2 doses, 3 doses gilt are attacked for 60 days and 90 days with HP-PRRSV-JXM-F5 respectively after immune Hit.Viremia virusemia occurs by attack gilt after 60 days, 10/10, average time is 25 days, and 8/10 gilt is dead, equally The gilt that water for injection instillation day after 90s is attacked, the sick toxaemia of 10/10 hair, 26 days average times, 9/10 small mother Pig is dead.

There are 10/10 generation PRRSV specific antibodies in 3 doses of intranasal immune gilt of instiling, the gilt for having 9/10 produces JX0708-F12 neutralizing antibody, antibody average titer 8.90 days after exempting from, after JX0708-F12 challenge infections, whole gilt There is viremia virusemia, time average out to 18 days, 8/10 gilt produces JX0708-F12 neutralizing antibodies, average neutralizing antibody drop 64,10/10 gilt is spent all to survive；120 days after exempting from, after JX0708-F12 infection attacks, viremia virusemia average time is 19 My god, 6/10 gilt neutralizing antibody level remains at 16, still positive for neutralizing antibody, the survival of 8/10 gilt；150 days after exempting from The gilt of poison group is attacked with JX0708-F12 infection, 10/10 has viremia virusemia, and average time is 20 days, 4/10 small mother Pig has neutralizing antibody detection, the survival of the gilt of average titer 16,5/10.

3 doses of intranasal instillation are immune, gm allotype PRRSV QH-08 are attacked in the gilt of poison, and 3 doses of instillation of completion are immunized small Pig all generates specific PRRSV antibody, has 4/10 piggy to generate to QH-08 neutralizing antibodies, average titer 8.3 doses of intranasal drops Attacked after note is immune 60 days in malicious group, 9/10 gilt has viremia virusemia, and average time is 16 days, and 2/10 gilt is dead Die, 5/10 gilt produces neutralizing antibody, average titer 64.Poison infection QH-08 is attacked within 120 days, it is in 6/10 gilt and anti- Body level remains at 32, viremia virusemia time of origin average out to 18 days, and 2/10 gilt is dead.QH-08 senses in 150 days after immune After dye attacks poison, 4/10 gilt produces neutralizing antibody.5/10 gilt is dead.

3 doses of intranasal instillation vaccinogen liquids, HP-PRRSV-JXM-F5 infection are attacked in the gilt of poison, have 2/10 to generate spy Different in nature PRRSV antibody, neutralizing antibody generation is had no, is attacked in malicious group within 30 days, 10/10 gilt has viremia virusemia, average Time is 27 days, and 8/10 gilt is dead；

Commercialization 3 doses of intranasal vaccinations of inactivation PRRSV vaccines are immune, HP-PRRSV-JXM-F5 infection is attacked in the gilt of poison, have 1/10 pig generates specific PRRSV antibody, and no HP-PRRSV-JXM-F5 neutralizing antibodies are attacked in malicious group for detectable 30 days, 10/10 gilt has viremia virusemia, and average time is 26 days, and 6/10 gilt is dead；

The effect of 3 doses of Nasal immunizations is identical with 2 doses of Nasal immunization effects, and there was no significant difference.2 doses, 3 doses of Nasal immunizations attack Comparing difference is notable two-by-two for viremia virusemia, clinical symptoms and the metainfective death toll and water for injection group of pig after poison.

Intranasal instillation vaccinogen liquid immune group immune effect and 3 doses of Nasal immunization group immune effects and 3 doses of Nasal immunizations In terms of specific PRRSV antibody tormations, induction neutralizing antibody after group is immune, significant difference；Different time attacks poison after immune, disease Number occurs for toxaemia, neutralizing antibody produces and attacked dead number significant difference after poison.

According to result above, 2 doses and 3 doses instil it is immune, invention vaccine to gilt immune period at 90-120 days, After 150 days, the piglet for infecting death substantially rises, therefore the vaccine of the invention, and route of inoculation instils for nasal cavity, and journey is immunized Sequence is 3 doses, is spaced 2 weeks, completes within one month immune.The immune period phase is 120 days.

3 doses of immune gilt of vaccine of intranasal immune invention of instiling, can blood serum induced neutralizing antibody generation, it is numerous to reduce pig Grow and occur with breathing syndrome virus virus infection mass formed by blood stasis, reduce gilt infection death, homogenic type PRRSV infection is drawn The Death prevention rate risen is 80%, and the protective rate that death is caused to special-shaped PRRSV infection is 60%, immune period 120 My god.

One group of intranasal immune 3 doses of invention vaccine that instil (amount of antigen and stablize 10 times of gilt of adjuvant complex components amount Dosage, volume are 5 milliliters) specific PRRSV antibody is all generated, there is 2/10 sow to generate in JX0708-F12 and anti- Body, average titer 32.After JX0708-F12 attacks poison after immune 60 days, 6/10 sow has viremia virusemia, and average time is 16 days, 10/10 sow survival, 6/10 sow produced JX0708-F12 neutralizing antibodies, average titer 64.Attack poison within 120 days JX0708-F12 is infected, 6/10 sow neutralizing antibody level is maintained at 32, and having 6/10 sow, there occurs viremia virusemia, time to put down It it is 17 days, no sow is dead.After poison is attacked in JX0708-F12 infection in 150 days after immune, 4/10 sow produces neutralizing antibody.1/ 10 sows are dead.

The sow 10 that two groups of ultrasound detection confirmations have been pregnant, invented in intranasal 3 doses of the instillation in 30-45 days of period of pregnancy Vaccine, 2 weeks after the completion of being immunized, 10 farrowing sows all generate specific PRRSV antibody, have 3/10 sow pig to generate pair HP-PRRSV-JXM-F5 neutralizing antibodies, average titer 64.HP-PRRSV-JXM-F5 is attacked in malicious group within 60 days, and 5/10 sow has Viremia virusemia occurs, and average time is 18 days, and 10/10 sow survival, 6/10 sow is produced in HP-PRRSV-JXM-F5 With antibody, average titer 128.Poison infection HP-PRRSV-JXM-F5 is attacked within 120 days, 6/10 sow neutralizing antibody level is maintained at 128, having 5/10 sow, there occurs viremia virusemia, time average out to 18 days, no sow are dead.150 days HP- after immune After poison is attacked in PRRSV-JXM-F5 infection, 3/10 sow produces HP-PRRSV-JXM-F5 neutralizing antibodies, and 1/10 sow is dead, and 8/ 10 sow has viremia virusemia, and the average virus mass formed by blood stasis time is 20 days.

Three groups with water for injection, at 0,14,28 day, 10 farrowing sows without PRRSV antibody were immunized in intranasal instil respectively, and 10 Head farrowing sow all detects without specific PRRSV antibody, is also detected without neutralizing antibody, is feminine gender, the 30 days use after immune QH-08 attacks malicious infection, has 1/10 sow to generate to QH-08 neutralizing antibodies, titre 4；10/10 sow generates viral blood Disease, viremia virusemia duration average out to 25 days, 9/10 has fervescence or death, and QH-08 is attacked in malicious group within 60 days, 10/10 Sow has viremia virusemia, and average time is 25 days, and 8/10 sow is dead or has symptom, and 1/10 sow produces QH-08 Neutralizing antibody, titre 8.Poison infection QH-08 is attacked within 120 days, 1/10 sow has neutralizing antibody, titre 8, there is 10/10 sow There occurs viremia virusemia, time average out to 23 days, 10/10 sow is dead or has symptoms of respiratory disease, body temperature rise.After immune After poison is attacked in QH-08 infection in 150 days, 10/10 sow has viremia virusemia.9/10 sow has body temperature rise and dead, 8/10 mother Pig has viremia virusemia, and the average virus mass formed by blood stasis time is 24 days.

One group and two groups of result and three groups of results contrasts, one group and three groups, two groups and three groups are immunized, attack malicious difference on effect Significantly.3 vaccinating agents intranasal instil immune sow and farrowing sow, wherein complete the pig of 3 doses of intranasal immunisations in period of gestation, i.e., two Group and three groups in terms of immune effect and disease symptomses protective rate difference it is not notable.Therefore the intranasal immune bosom of instiling of the vaccine of invention Pregnant sow and sow are safely, effectively.When invention vaccine is reduced viremia virusemia to gm allotype PRRSV JX0708-F12 infection Between, reduce dead or morbidity effect, it is 70% to reduce morbidity and dead effect.

Indicated above, vaccine of the invention can make 2 kinds of specifications, and a specification uses gilt, and route of inoculation is intranasal drop Nose, it is spaced 2 weeks, is inoculated with three times in January, immune period is 120 days, and a specification is used in sow and farrowing sow, inoculation way Footpath is intranasal collunarium, is spaced 2 weeks, is inoculated with three times in January, and immune period is 120 days.

The PRRSV-KPWV-SwIVA-RV vaccine anti-swine flu effects of embodiment 5 and effect

Method：

5.1. experimental design is collected with sample

Health, confirmation are without SwIAV H1N1 and H1N2 hemagglutination inhibition antibody piglets (n=40), and selected piglet immunological program is such as Embodiment 4, secondary Nasal immunization was completed in one month, is divided into 4 test groups (n=10 heads/group) by vaccine formulation and difference, 1st group is that PBS simulates Nasal immunization pig, does not attack poison；2nd group is that PBS simulates Nasal immunization pig.Attack poison；3rd group is PRRSVKPWV-SwIVA-RV immune swines, attack poison；4th group is the stable Adjuvanted vaccines immune swines of PRRSVKPWV-SwIVA-RV+, Poison is attacked, the experimental animal welfare method raising formulated by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences and test operation.

Each immunizing dose such as embodiment 4, H1N2 amount of antigen is inactivated as before inactivation 10 in vaccine⁷TCID₅₀H1N2, vaccine Volume is 5 milliliters.Attack poison is the relatively strong heterologous SwIAV H1N1 2mL (6 × 10 of virulence⁶TCID50/2 mL), 1mL instills nose Interior, 1mL instills tracheae.Plasma sample is taken in vaccinogen liquid, vaccine inoculation or during postmortem.Anus temperature is detected daily after attacking poison Degree, Nasopharyngeal swabs immersion 2mL is collected within 4 days after attacking poison, 6 days (DPC) anaesthetizes pig after poison is attacked, and detects damage and the marking of lung.

BAL fluid (Broncho-alveolar lavage, BAL) is collected to titrate for virus titer；Receive Collection right upper lung leaf texture 1g is suspended in 3mL DMEM, after being homogenized homogeneous, collects supernatant and is used to detect antibody response.Lung tissue good fortune Your Malin, which fixes, carries out histopathology and immunochemistry assessment.In attack, 0 used density gradient media lymph with 6 days after attack Cell prepares SepMate-50 pipes (being purchased from Canadian Stemcell companies) separation periphery monokaryon lymphocyte and carries out lymphocyte Propagation and flow cytometry.

5.2 cells are bred and flow cytometry analysis

SwIAV antigen-specific lymphocyte proliferation assays PBMCs, the liquid phase non-radioactive proliferation assay reagent of cell titer 96 Box (being purchased from Promega companies of the U.S.) provides operating guidance by producer and carried out.The sterile orifice plate of U-shaped bottom 96 is inoculated with 1,000,000 years per hole Cell, same two holes, stimulated or nutrient solution is control to live SwIAV H1N2 of 0.1MOI, 37 DEG C, be incubated 72 under the conditions of 5% Hour, add MTS+PMS solution reactions 4 hours, reading is detected at ELISA plate readers OD490nm.Stimulus index (Stimulation index, SI) is to stimulate peripheral mononuclear cells (PBMCs) the OD divided by control cell OD of same pig.DPC The PBMCs flow cytometries of separation in 0 day determine different T cell subgroups.6 days (DPC 6) separates PBMCs and used after poison is attacked 0.1MOISwIAV H1N2 or H1N1 are stimulated 72 hours, and activation (IFN γ is determined with flow cytometry analysis after immunostaining⁺)T The frequency of cell subsets.Anti- pig CD3 ε, anti-pig CD4 α, the anti-anti- pig CD8 β of pig CD8 α, anti-pig δ-chain, anti-porcine IFN γ are (purchased from U.S. BD biosciences companies of state).

5.3 virus titers detect

Nose swab, BAL fluid are serially diluted again with trypsase containing TPCK- (1 μ g/mL) DMEM 10.Move Enter to be paved with the orifice plate of mdck cell 96, in 37 DEG C, 5%CO₂Cultivated 48 hours in incubator, influenza A virus NP albumen monoclonal antibody conducts First antibody (#M058, purchased from CalBioreagents companies of the U.S.), it is anti-that AlexaFluor 488 combines sheep anti-mouse igg (H+L) Body (being purchased from Life technologies companies of the U.S.) is used as secondary antibody, uses fluorescence microscope (Japanese Olympus companies Product) record fluorescence, virus infection titer is calculated using Reed and Muench methods.

5.4 antibody titers detect

All Swine serums (H1) damage enzyme (Receptor destroying with 20% high woods soil or H3 serum acceptor Enzyme RDE) processing.50% chicken red blood cell removes non-specific suppression processing.All serum dilute for 5 times all with PBS, by OIE Handbook designation method determines four representative swine flu blood clottings and suppressed.

μ l of 8HA units/50,1% chicken RBCs are used to titrate.Based on HI antibody male rotaries and HI titres during 4 times of titre, HI Titre 1：40 be protection antibody.

5.5 serum neutralizing antibodies detect

All 56 DEG C of inactivation 30min of serum, serum：MEM=1：After 5 dilutions, used on 96 orifice plates and contain 3 μ g/ml TCPK pancreases Protease (being purchased from Sigma-Aldrich) MEM 2 is serially diluted again, swine influenza virus

Reacted in 100TCID50 adding holes, move into 37 DEG C of mdck cell plate, 5%CO2 and be incubated 1h, neutralize 50% completely Serum highest dilution be neutralizing antibody titers.Cell is fixed with 4% formalin solution, uses first stream virus nucleoprotein list Anti-, horseradish peroxidase combination rabbit anti-mouse igg, AEC (chromogenaminoethylcarbazole) substrate are (purchased from U.S. Power ＆ light company of state) detection viral antigen.Neutralizing antibody titers are more than or equal to 1:40 be aversion response.

The undiluted nose swab of each group and 1:200 dilution BAL liquid, serum, the IgG in lung and IgA reactions are compared, each group Pig BAL liquid neutralizing antibodies are detected and compared.

5.6 pathology and SABC detection

5 days after challenge infection, all pigs anaesthetize postmortem, the more focus mottled-tan of lung macroscopic damage feature and solidification, note Record as percentage.Collect at the top of lung, fixed close to center portion, diaphragm portion lobe of the lung tissue with 10% formalin, be embedded in solid 4 μm of slabs are cut into paraffin and are used for pathological analysis and haematine, eosin stains.Injury severity score is with damage profile and outside All monocyte (PMN) infiltrations divide 0-3 levels.Viral antigen is used on lung's SwIAV specific antigens and air flue epidermal cell ImmunohistochemistryMethods Methods detect：After the dyeing of SwIAV nucleoprotein specific antibody, by the guide VECTASTAIN of manufacturer's offer Elite ABC reagents (#PK-7100, purchased from U.S. Vector Labs) processing shows signal.

5.7 statistics

Every group of data represent that HI and VN titres are represented with geometric mean titer ± 95%CI with average.Virus titer is done Logarithm process and analysis, (being purchased from GraphPad Software companies of the U.S.) is carried out non-on the softwares of GraphPad Prism 5 Parameter Kruskal-Wallis carries out Dunn ' s post hoc inspections and compared after examining, P<0.05 is statistically notable.

As a result：

1.PRRSV-KPWV-SwIVA-RV physical features

The most of PRRSVKPWV-SwIVA-RV forms of scanning electron microscopic observation are circle, granular size in 200-300 nm, A diameter of 235 ± the 50nm of grating confirmation PRRSV-KPWVSwIVA-RV reconstruct Virosome particles.Inactivation purifying PRRS totivirus with SwIAV H1N2 form virion efficiency 60%.

2. the cell and humoral immune reaction of two Nasal immunization inductions

PBMCs is separated from 30 days (DPV90/DPC4) days after immune swine, is pierced with vaccine strain virus (SwIAV H1N2) Swash, lymphopoiesis is assessed:The average thorn of the stable adjuvant compound vaccine Nasal immunization pigs of PRRSVKPWV-SwIVA-RV+ It is 2.3 ± 0.8 to swash index, the mean stimulation indices of independent PRRSVKPWV-SwIVA-RV antigens Nasal immunization pig for 1.51 ± 0.5, physiological saline simulation Nasal immunization pig mean stimulation indices are 0.75 ± 0.9.

The stimulus index of 3 immune swines of vaccine is significantly higher than physiological saline simulation Nasal immunization pig mean stimulation indices (p ＜ 0.001.The stimulus index of 3 immune swines of vaccine is significantly higher than independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization pig Stimulus index (p ＜ 0.01.The secondary Nasal immunization pig Th/Tm (CD3 of vaccine⁺CD4⁺CD8αα⁺) frequency be 9.2 ± 0.6%, it is single Only PRRSVKPWV-SWIVA-RV antigen Nasal immunization pig Th/Tm (CD3⁺CD4⁺CD8αα⁺) average frequency 6.5 ± 1.8%, it is raw Manage salt water modeling Nasal immunization pig Th/Tm (CD3⁺CD4⁺CD8αα⁺) average frequency 5.3 ± 1.0%.The secondary Nasal immunization of vaccine Pig Th/Tm (CD3⁺CD4⁺CD8αα⁺) frequency be significantly higher than physiological saline simulation Nasal immunization pig Th/Tm (CD3⁺CD4⁺CD8α α⁺) frequency (p ＜ 0.01.Illustrate the effect of the inducing cellular immune of stable adjuvant compound.Independent PRRSV-KPWV-SWIVA- RV antigen Nasal immunization pig Th/Tm (CD3⁺CD4⁺CD8αα⁺) frequency and simulation immune swine Th/Tm (CD3⁺CD4⁺CD8αα⁺) without aobvious Write difference (p ＜ 0.05.

Secondary Nasal immunization pig lymphocyte poison cell (the CTLs) (CD3 of vaccine⁺CD4^-CD8aβ⁺) frequency averaging be 11.8 ± 3.6%, independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization pig lymphocyte poison cell (CTLs) (CD3⁺CD4^- CD8aβ⁺) frequency averaging be 8.1 ± 2.6%, physiological saline simulation Nasal immunization pig lymphocyte poison cell (CTLs) (CD3⁺ CD4^-CD aβ⁺) frequency averaging be 7.5 ± 2.1%.Secondary Nasal immunization pig lymphocyte poison cell (the CTLs) (CD3 of vaccine⁺ CD4^-CD8aβ⁺) frequency be significantly higher than physiological saline simulation Nasal immunization pig lymphocyte poison cell (CTLs) (CD3⁺CD4^-CD aβ⁺) frequency (p ＜ 0.01.Illustrate vaccine and stablize adjuvant induction CTL effect.

Attack within 30 days after the secondary Nasal immunization of vaccine malicious DPC 0, vaccine immunity Swine plasma HI titres average 29.5 ± 14, individually PRRSVKPWV-SWIVA-RV antigen Nasal immunization Swine plasmas HI titre average 11.9 ± 4.7, and physiological saline simulation collunarium Immune Swine plasma HI titres average 2.5 ± 0.1, vaccine immunity pig, independent PRRSV-KPWV-SWIVA-RV antigens Nasal immunization Pig HI antibody levels are all remarkably higher than immune pig (the vaccine immunity pigs of p ＜ 0.001, p ＜ 0.01, the independent PRRSVKPWV- of simulation SWIVA-RV antigen Nasal immunization pig HI antibody level significant differences.Illustrate that stable adjuvant compound has enhancing specific antibody The effect of generation.Also illustrate vaccine antigen and stablize the ability that adjuvant compound has inducing specific humoral response.

Attack malicious DPC 4 within 30 days after the secondary Nasal immunization of vaccine, malicious (H1N2), vaccine immunity pig blood are attacked using vaccine strain virus Starch IgG OD450nm average 1.7 ± 1.3, independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization Swine plasma IgG OD450nm Average 0.8 ± 1.6, and physiological saline simulation Nasal immunization Swine plasma IgG OD450nm average 0.3 ± 0.7.Vaccine immunity pig, Independent PRRSV-KPWV-SWIVA-RV antigen Nasal immunization Swine plasma IgG levels are all remarkably higher than immune pig (the p ＜ of simulation 0.001.Vaccine immunity pig, the horizontal significant differences of independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization Swine plasma IgG (p ＜ 0.01.Illustrate that stable adjuvant compound has enhancing specific antibody nucleus formation.Also illustrate vaccine antigen and stablize adjuvant and answer Compound has the ability of inducing specific humoral response.

30 days after the secondary Nasal immunization of vaccine, poison, vaccine immunity Swine plasma IgG are attacked using heterologous swine influenza virus OD450nm average 0.9 ± 0.5, independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization Swine plasma IgG OD450nm are averaged 0.6 ± 0.4, and physiological saline simulation Nasal immunization Swine plasma IgG OD450nm average 0.2 ± 0.5.Vaccine immunity pig, individually PRRSVKPWV-SWIVA-RV antigen Nasal immunization Swine plasma IgG levels are significantly higher than the immune pig of simulation.Vaccine immunity pig, list The horizontal significant differences of only PRRSVKPWV-SWIVA-RV antigen Nasal immunization Swine plasma IgG.Illustrate that stable adjuvant compound has Strengthen specific antibody nucleus formation.Also illustrate vaccine antigen and stablize adjuvant induction to heterologous swine influenza virus specificity humoral The ability of reaction.

3. clinical case changes

The body temperature of pig is that 104 ℉ think to have a fever.Simulation Immunization pig and porcine reproductive and respiratory syndrome inactivation have purified The fever course of disease of whole viral antigen-pig first stream virus reconstruct virion (PRRSKPWV-SwIVA-RV) Nasal immunization pig is 1-4 My god, the vaccine Nasal immunization pig heating course of disease 1 day.

Simulate Nasal immunization and attack malicious pig：First day ℉ of body temperature average out to 105.2 ± 0.4 after poison is attacked, is attacked after poison second day The ℉ of body temperature average out to 104.2 ± 0.4, second day ℉ of body temperature average out to 104.8 ± 0.4 after poison is attacked, the 4th day body temperature is put down after attacking poison It is 104.2 ± 0.4 ℉, attacks the 5th day ℉ of body temperature average out to 103.6 ± 0.4 after poison, attacks the 6th day body temperature average out to after poison 102.8±0.2℉。

PRRSKPWV-SwIVA-RV Immunization pigs：First day ℉ of body temperature average out to 104.6 ± 0.2 after poison is attacked, after attacking poison Second day ℉ of body temperature average out to 103.8 ± 0.2, second day ℉ of body temperature average out to 104.2 ± 0.6 after poison is attacked, is attacked after poison the 4th day The ℉ of body temperature average out to 104.4 ± 0.8, the 5th day ℉ of body temperature average out to 103.4 ± 0.4 after poison is attacked, the 6th day body temperature is put down after attacking poison It is 102.6 ± 0.4 ℉.

Vaccine Nasal immunization attacks malicious pig：First day ℉ of body temperature average out to 104.2 ± 0.2 after poison is attacked, attacks poison body after second day The warm ℉ of average out to 103.6 ± 0.2, attack poison second day after the ℉ of body temperature average out to 102.8 ± 0.1, attack poison the 4th day after body temperature be averaged For 102.4 ± 0.1 ℉, the poison ℉ of body temperature average out to 102.5 ± 0.1, body temperature average out to 102 ± 0.1 after the 6th day after the 5th day are attacked ℉。

Simulate Nasal immunization and do not attack malicious pig：The 0th day ℉ of body temperature average out to 102.2 ± 0.2 after poison is attacked, is attacked after poison first day The ℉ of body temperature average out to 102.8 ± 0.1, attack second day ℉ of body temperature average out to 104.8 ± 0.4 after poison, attack poison the 4th day after body temperature put down Be 104.2 ± 0.4 ℉, attack poison the 5th day after the ℉ of body temperature average out to 103.6 ± 0.4, body temperature average out to 102.8 after the 6th day ±0.2℉。

PRRSKPWV-SwIVA-RV Immunizations temperature of pig body attacks malicious temperature of pig body without significant difference (p ＜ with vaccine Nasal immunization 0.05.Although macroscopical injury of lungs score value also (p ＜ 0.05, is directed to the small local damage of lung without significant difference.Attack after poison 6 days Simulation Nasal immunization is consolidated with malicious pig lung solidification average out to 22.6 ± 15.2%, PRRSKPWV-SwIVA-RV Immunization pig lungs is attacked Change average out to 23.1 ± 7.5%, vaccine Nasal immunization attacks malicious pig lung solidification average out to 17.3 ± 16.5%.Illustrate that vaccine reduces pig Pulmonary lesion caused by influenza virus.

Nasal immunizations are simulated within 6 days after attacking poison and attack malicious pig pneumonia disease (H＆E) score value average out to 1.3 ± 1.6, PRRSKPWV- SwIVA-RV Immunization pig pneumonia disease (H＆E) score values average out to 1.4 ± 0.2, vaccine Nasal immunization attacks malicious pig pneumonia disease (H＆E) Score value average out to 1.6 ± 0.2, physiological saline simulation is immune and does not attack malicious pig pneumonia disease (H＆E) score value average out to 0.2 ± 0.1.

SwIAV antigen immune group score values：Attack after poison 6 days, simulation Nasal immunization and attack malicious swine flue antigen reaction Mild-natured is 1.9 ± 1.7, PRRSKPWV-SwIVA-RV Immunization swine flue antigen reactivity average out to 1.5 ± 0.6, Vaccine Nasal immunization attacks malicious swine flue antigen reactivity average out to 0.1 ± 0.5, and physiological saline simulation is immune and does not attack malicious pig Influenza antigen reactivity average out to 0.1 ± 0.6.Vaccine Nasal immunization attacks malicious swine flue antigen reactivity and simulation drop Nose, which is immunized and attacked malicious swine flue antigen reactivity and is relatively substantially reduced, (p ＜ 0.01, illustrates that vaccine causes the anti-first of lung Protective immunity is flowed, lung virus substantially reduces, and vaccine reduces pulmonary infection.

4 days after infection attack, the influenza virus A porcine titre of nose swab：Simulate Nasal immunization and attack malicious hog snout swab pig Influenza A virus titre TCID50/mL (log10) average out to 5.9 ± 0.1, PRRSKPWV-SwIVA-RV Immunization hog snouts Swab influenza virus A porcine titre TCID50/mL (log10) average out to 6.1 ± 0.1, vaccine Nasal immunization attacks malicious hog snout swab Influenza virus A porcine titre TCID50/mL (log10) average out to 4.3 ± 0.1.It is consistent with lung SABC score value, attacking poison Afterwards the 4th day, virus than independent PRRSKPWV-SwIVA-RV Immunizations, simulation Nasal immunization and was attacked in vaccine immunity hog snout Viral low 6-8 times of malicious hog snout.

Attack after poison 6 days, simulation Nasal immunization and attack influenza virus A porcine in malicious hog snout bronchoalveolar lavage system liquid (BAL) Titre TCID50/mL (log10) average out to 5.7 ± 1.1, PRRSKPWV-SwIVA-RV Immunization hog snout bronchoalveolar lavages It is influenza virus A porcine titre TCID50/mL (log10) average out to 3.1 ± 0.8 in liquid (BAL), vaccine Nasal immunization attacks poison Influenza virus A porcine titre TCID50/mL (log10) average out to 3.3 ± 2 in hog snout bronchoalveolar lavage system liquid (BAL).With Simulate Nasal immunization and attack virus titer in malicious hog snout bronchoalveolar lavage system liquid (BAL) and compare, PRRSKPWV-SwIVA-RV Virus and vaccine Nasal immunization attack pig first in malicious hog snout bronchoalveolar lavage system liquid (BAL) in Immunization hog snout bronchovesicular Type influenza virus declines 40-37 times respectively, is disclosed in virus replication in the ventilation epidermal cell of immune swine or is scattered seldom.Say The effect of bright vaccine mucosal immunity.

4. anamnesis IFN γ secretion lymphocyte reaction is activated after swine influenza virus attack in blood

Anamnesis IFN γ secretion lymphocyte reaction is activated after swine influenza virus is attacked in blood to assess, separation PBMCs stimulates assessment activation IFN γ lymphocyte sub- with vaccine strain virus H1N2, the malicious H1N1SwIAV virions of attack respectively Group.

H1N2 is stimulated, and is simulated Nasal immunization and is attacked malicious pig CD3⁺IFNγ⁺Cell accounting average out to 6.2 ± 1.8%, PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺IFNγ⁺Cell accounting average out to 5.0 ± 0.7%, vaccine Nasal immunization attacks poison Pig CD3⁺IFNγ⁺Cell accounting average out to 8.1 ± 5.1%, physiological saline simulation is immune and does not attack malicious pig CD3⁺IFNγ⁺Cell Accounting average out to 3.6 ± 1.3%.Vaccine Nasal immunization attacks malicious pig CD3⁺IFNγ⁺Cell accounting is higher than simulation Nasal immunization and attacks Malicious pig CD3⁺IFNγ⁺(p ＜ 0.05, vaccine Nasal immunization attack malicious pig CD3 to cell accounting⁺IFNγ⁺Cell accounting is higher than PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺IFNγ⁺(p ＜ 0.05, vaccine Nasal immunization attack malicious pig CD3 to cell accounting⁺ IFNγ⁺Cell accounting is immune higher than physiological saline simulation and does not attack malicious pig CD3⁺IFNγ⁺Cell accounting (p ＜ 0.05, but without aobvious Write difference.

H1N1 is stimulated, and is simulated Nasal immunization and is attacked malicious pig CD3⁺IFNγ⁺Cell accounting average out to 6.3 ± 5.1%, PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺IFNγ⁺Cell accounting average out to 4.9 ± 4.1%, vaccine Nasal immunization attacks poison Pig pig CD3⁺IFNγ⁺Cell accounting average out to 9.6 ± 5.3%, physiological saline simulation is immune and does not attack malicious pig CD3⁺IFNγ⁺Carefully Born of the same parents' accounting average out to 3.9 ± 1.3%.Vaccine Nasal immunization attacks malicious pig CD3⁺IFNγ⁺Cell accounting is higher than PRRSKPWV- SwIVA-RV Immunization pigs CD3⁺IFNγ⁺(p ＜ 0.05, vaccine Nasal immunization attack malicious pig CD3 to cell accounting⁺IFNγ⁺Cell Accounting is higher than simulation Nasal immunization and attacks malicious pig CD3⁺IFNγ⁺Cell accounting (p ＜ 0.0, but without significant difference.

No matter H1N1, H1N2 are stimulated, invention vaccine (the stable adjuvant compounds of PRRSKPWV-SwIVA-RV+) immune swine ratio The independent immune swine activation CD3 of PRRSKPWV-SwIVA-RV⁺IFNγ⁺The frequency of cell significantly improves.

H1N2 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3^-IFNγ⁺Cell accounting average out to 0.7 ± 0.1%, Simulate Nasal immunization and attack malicious pig CD3^-IFNγ⁺Cell accounting average out to 0.41 ± 0.20%, PRRSKPWV-SwIVA-RV exempts from Epidemic disease attacks malicious pig CD3^-IFNγ⁺Cell accounting average out to 0.81 ± 1.9%, vaccine Nasal immunization attack malicious pig CD3^-IFNγ⁺Cell accounts for Than average out to 0.3 ± 0.1%.Vaccine Nasal immunization attacks malicious pig CD3^-IFNγ⁺Cell accounting is substantially less than PRRSKPWV-SwIVA- RV Immunization pigs CD3^-IFNγ⁺(p ＜ 0.001, physiological saline simulation is immune and attacks malicious pig CD3 for cell accounting^-IFNγ⁺Carefully Born of the same parents' accounting, which is substantially less than, to be simulated Nasal immunization and attacks malicious pig CD3^-IFNγ⁺Cell accounts for (p ＜ 0.01H1N1 stimulations, physiological saline mould Intend immune and do not attack malicious pig CD3^-IFNγ⁺Cell accounting average out to 0.8 ± 0.2%.Simulate Nasal immunization and attack malicious pig CD3^-IFN γ⁺Cell accounting average out to 0.5 ± 0.30%, PRRSKPWV-SwIVA-RV Immunization pigs CD3^-IFNγ⁺Cell accounting is put down It is 1.8 ± 1.4%, vaccine Nasal immunization attacks malicious pig CD3^-IFNγ⁺Cell accounting is substantially less than PRRSKPWV-SwIVA- RV Immunization pig CD3^-IFNγ⁺(p ＜ 0.001, physiological saline simulation is immune and does not attack malicious pig CD3 for cell accounting^-IFNγ⁺Carefully Born of the same parents' accounting, which is substantially less than, to be simulated Nasal immunization and attacks malicious pig CD3^-IFNγ⁺Cell accounts for (p ＜ 0.01).No matter H1N1, H1N2 are stimulated, In CD3 in the independent immune swines of PRRSKPWV-SwIVA-RV^-IFNγ⁺Non- T is significantly higher than invention vaccine (PRRSKPWV-SwIVA-RV + stable adjuvant compound) in immune swine CD3^-IFNγ⁺Non-T cell.

H1N2 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting average out to 0.2 ± 0.2%, simulate Nasal immunization and attack malicious pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting average out to 0.5 ± 0.3%, PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting average out to 0.8 ± 0.4%, vaccine drop Nose Immunization pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting average out to 1.3 ± 0.6%.Vaccine Nasal immunization attacks malicious pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting is significantly higher than PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺CD4^-CD8αβ⁺ IFNγ⁺Cell accounting (p ＜ 0.01, also significantly greater than simulates Nasal immunization and attacks malicious pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounts for Than (p ＜ 0.01, also significantly greater than physiological saline simulation are immune and do not attack malicious pig CD3⁺CD4^-CD8αβ⁺IFNγ⁺Cell accounting (p ＜ 0.01

No matter H1N1, H1N2 are stimulated, invention vaccine (PRRSKPWV-SwIVA-RV+ stable adjuvant compound) immune swine and The independent immune swines of PRRSKPWV-SwIVA-RV can make activation CTL (CD3 compared with compareing pig with simulation⁺CD4^-CD8αβ⁺IFN γ⁺) cell frequencies significantly raise.

H1N2 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.0 ± 0.1%, simulate Nasal immunization and attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.8 ± 2.2%, PRRSKPWV- SwIVA-RV Immunization pigs CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.1 ± 0.2%, vaccine Nasal immunization attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 2.4 ± 1.3%.Vaccine Nasal immunization attacks malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting shows Work is higher than PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺CD4⁺ IFNγ⁺Cell accounting (p ＜ 0.01, also significantly greater than mould Intend Nasal immunization and attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting (p ＜ 0.001, also significantly greater than physiological saline simulation it is immune and Malicious pig CD3 is not attacked⁺CD4⁺IFNγ⁺Cell accounting (p ＜ 0.001,

H1N1 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.0 ± 0.1%, simulate Nasal immunization and attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.8 ± 2.2%, PRRSKPWV- SwIVA-RV Immunization pigs CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 1.1 ± 0.2%, vaccine Nasal immunization attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting average out to 2.4 ± 1.3%.Vaccine Nasal immunization attacks malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting shows Work is higher than PRRSKPWV-SwIVA-RV Immunization pigs CD3⁺CD4⁺ IFNγ⁺Cell accounting (p ＜ 0.01, also significantly greater than mould Intend Nasal immunization and attack malicious pig CD3⁺CD4⁺IFNγ⁺Cell accounting (p ＜ 0.001, also significantly greater than physiological saline simulation it is immune and Malicious pig CD3 is not attacked⁺CD4⁺IFNγ⁺Cell accounting (p ＜ 0.001,

No matter H1N1, H1N2 are stimulated, invention vaccine (the stable adjuvant compounds of PRRSKPWV-SwIVA-RV+) immune swine ratio The independent immune swine activation CD3 of PRRSKPWV-SwIVA-RV⁺CD4⁺IFNγ⁺Cell frequencies significantly improve.

H1N2 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.2 ± 0.2%, simulate Nasal immunization and attack malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.1 ± 0.1%, PRRSKPWV-SwIVA-RV Immunization pigs CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.4 ± 0.7%, vaccine drop Nose Immunization pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.08 ± 0.01%.Vaccine Nasal immunization attacks malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting is substantially less than PRRSKPWV-SwIVA-RV Immunization pigs CD3^-CD4^-CD8α⁺IFN γ⁺Cell accounting (p ＜ 0.001.PRRSKPWV-SwIVA-RV Immunization pigs CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting is high Malicious pig CD3 is attacked in simulating Nasal immunization and attacking malicious pig vaccine Nasal immunization^-CD4^-CD8α⁺IFNγ⁺Cell accounting (p ＜ 0.05, It is immunized higher than physiological saline simulation and does not attack malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting (p ＜ 0.05,

H1N1 is stimulated, and physiological saline simulation is immune and does not attack malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.3 ± 0.3%, simulate Nasal immunization and attack malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.3 ± 0.4%, PRRSKPWV-SwIVA-RV Immunization pigs CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.7 ± 0.6%, vaccine drop Nose Immunization pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting average out to 0.07 ± 0.01%.Vaccine Nasal immunization attacks malicious pig CD3^-CD4^-CD8α⁺IFNγ⁺Cell accounting is substantially less than PRRSKPWV-SwIVA-RV Immunization pigs CD3^-CD4^-CD8α⁺IFN γ⁺Cell accounting (p ＜ 0.001.

No matter H1N1, H1N2 are stimulated, in CD3 in the independent immune swines of PRRSKPWV-SwIVA-RV^-CD4^-CD8α⁺IFNγ⁺ NK cell frequencies be significantly higher than in invention vaccine (PRRSKPWV-SwIVA-RV+ stable adjuvant compound) immune swine CD3^- CD4^-CD8α⁺IFNγ⁺NK cell frequencies.

5. the humoral immune reaction that immune swine is attacked after poison

Attack poison within 30 days after the secondary Nasal immunization of pig, the specific system of pig, local immunity reaction are detected and analyzed.

Hog snout swab Specific IgA antibody：Secondary vaccine Nasal immunization hog snout swab Specific IgA antibody is averaged OD450nm is 0.96 ± 0.30, and independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization hog snout swab Specific IgA antibody is put down Equal OD450nm is 0.47 ± 0.20, and the average OD450nm of physiological saline simulation immune swine nose swab Specific IgA antibody is 0.04 ± 0.20, it is 0.18 ± 0.22 only to attack the average OD450nm of malicious hog snout swab Specific IgA antibody unavoidably.Secondary vaccine collunarium is exempted from Epidemic disease hog snout swab Specific IgA antibody level is significantly higher than physiological saline simulation immune swine nose swab Specific IgA antibody (P ＜ 0.001, also significantly greater than independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization hog snout swab Specific IgA antibody is horizontal (P ＜ 0.01, malicious hog snout swab Specific IgA antibody level is also significantly greater than only attacked unavoidably and ((P ＜ 0.001, illustrates invention vaccine Reacted induction of local mucosal immunity, stable adjuvant compound can promote local mucous membrane specific antibody to generate.

IgA in lung lysate：IgA OD450nm average out to 1.52 in secondary vaccine Nasal immunization pig lung lysate ± 0.63, IgA OD450nm average out to 0.16 in independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization pig lung lysate ± 0.20, physiological saline simulation immune swine BAL liquid IgA OD450nm average out to 0.05 ± 0.23, only attacks malicious pig lung lysate unavoidably Middle IgA OD450nm average out to 0.25 ± 0.57.Secondary vaccine Nasal immunization pig lung lysate reclaimed water is flat to be significantly higher than physiology salt IgA levels (P ＜ 0.001, also significantly greater than independent PRRSVKPWV-SWIVA-RV antigens in Fluid Dynamics immune swine lung lysate P ＜ 0.001 in Nasal immunization pig lung lysate, IgA levels (P ＜ in malicious pig lung lysate are also significantly greater than only attacked unavoidably 0.001, illustrate that the vaccine-induced pig lung tissue of invention produces high-level IgA, stable adjuvant compound can strengthen mucous membrane local I gA Generation.

Swine plasma IgG antibody is reacted：The average OD450nm of secondary vaccine Nasal immunization Swine plasma specific IgG antibodies is 0.76 ± 0.4, the average OD450nm of independent PRRSV-KPWVSWIVA-RV antigens Nasal immunization Swine plasma specific IgG antibodies are 0.73 ± 0.5, the average OD450nm of physiological saline simulation immune swine plasma specific IgG antibody is 0.25. ± 0.7, unavoidably only It is 0.22 ± 1.1 to attack the average OD450nm of malicious Swine plasma specific IgG antibodies.Secondary vaccine Nasal immunization Swine plasma specific IgG Antibody level is significantly higher than physiological saline simulation immune swine nose swab specificity IgA antibody and (P ＜ 0.01, is also significantly greater than independent PRRSVKPWV-SWIVA-RV antigen Nasal immunization Swine plasmas specific IgG antibodies level (P ＜ 0.01, illustrates that invention vaccine lures Systemic humoral immune reaction is led, stable adjuvant compound has systemic characteristic antibody humidification.

Swine plasma blood clotting suppresses titre：Secondary vaccine Nasal immunization Swine plasma blood clotting suppresses titre average out to Log 2 (81 ± 75, independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization Swine plasma blood clotting suppress titre average out to Log 2 (63 ± 10), the immune Swine plasma blood clotting of physiological saline simulation suppresses titre average out to Log 2 (4.8 ± 3.5), only attacks malicious Swine plasma unavoidably Blood clotting suppresses titre average out to Log 2 (43.7 ± 31.9).It is significantly high that secondary vaccine Nasal immunization Swine plasma blood clotting suppresses titre Immune Swine plasma blood clotting, which is simulated, in physiological saline suppresses titre (P ＜ 0.01, also significantly greater than independent PRRSVKPWV-SWIVA-RV Antigen Nasal immunization Swine plasma blood clotting suppresses titre levels and (P ＜ 0.01, also significantly greater than only attacks malicious Swine plasma blood clotting suppression unavoidably Titre levels processed (P ＜ 0.01, illustrate the vaccine-induced high-level Swine plasma hemagglutination inhibition antibody of invention, stable adjuvant compound The generation of Swine plasma hemagglutination inhibition antibody can be strengthened.

BAL liquid IgA：Secondary vaccine Nasal immunization pig BAL liquid IgA OD450nm average out to 2.1 ± 0.38, individually PRRSVKPWV-SWIVA-RV antigen Nasal immunization pig BAL liquid IgA OD450nm average out to 0.41 ± 0.20, physiological saline simulation Immune swine BAL liquid IgA OD450nm average out to 0.21 ± 0.20, only attacks malicious pig BAL liquid IgA OD450nm average out to unavoidably 0.81±0.30.Secondary vaccine Nasal immunization pig BAL liquid IgA levels are significantly higher than physiological saline simulation immune swine BAL liquid IgA water Flat (P ＜ 0.001, also significantly greater than independent PRRSVKPWV-SWIVA-RV antigens Nasal immunization pig BAL liquid IgA levels (P ＜ 0.01, malicious pig BAL liquid IgA levels are also significantly greater than only attacked unavoidably (P ＜ 0.01, illustrates the vaccine-induced pig high level mucous membrane of invention Local I gA is horizontal, and stable adjuvant compound can strengthen mucous membrane local I gA generations.

BAL liquid IgG：Secondary vaccine Nasal immunization pig BAL liquid IgG OD450nm average out to 0.93 ± 0.84, individually PRRSVKPWV-SWIVA-RV antigen Nasal immunization pig BAL liquid IgG OD450nm average out to 0.15 ± 0.25, physiological saline simulation Immune swine BAL liquid IgG OD450nm average out to 0.18 ± 0.12, only attacks malicious pig BAL liquid IgG OD450nm average out to 0.49 unavoidably ±0.95.Secondary vaccine Nasal immunization pig BAL liquid IgG levels are significantly higher than physiological saline simulation immune swine BAL liquid IgG levels (P ＜ 0.001, also significantly greater than independent PRRSV-KPWV-SWIVA-RV antigens Nasal immunization pig BAL liquid IgA levels (P ＜ 0.001, malicious pig BAL liquid IgG levels are also significantly greater than only attacked unavoidably (P ＜ 0.01, illustrates the vaccine-induced pig high level mucous membrane of invention Local I gG is horizontal, and stable adjuvant compound can strengthen mucous membrane local I gG generations.

BAL liquid neutralizing antibodies：Secondary vaccine Nasal immunization pig BAL liquid neutralizing antibody average out to Log 2 (64.5 ± 25) are single Only PRRSVKPWV-SWIVA-RV antigen Nasal immunization pig BAL liquid neutralizing antibody average out to Log 2 (27.3 ± 21.6), physiology Salt water modeling immune swine BAL liquid neutralizing antibody average out to Log 2 (0.12 ± 0.1), malicious pig BAL liquid neutralizing antibodies are only attacked unavoidably and are put down It is Log 2 (34.5 ± 35.6).It is immune that secondary vaccine Nasal immunization pig BAL liquid IgG levels are significantly higher than physiological saline simulation Pig BAL liquid IgG level (P ＜ 0.001, also significantly greater than independent PRRSV-KPWV-SWIVA-RV antigens Nasal immunization pig BAL liquid IgA is horizontal (P ＜ 0.001, also significantly greater than only to be attacked malicious pig BAL liquid IgG levels and (P ＜ 0.01, illustrates that invention is vaccine-induced unavoidably Pig lung tissue high level neutralizing antibody, stable adjuvant compound can strengthen the generation of lung tissue neutralizing antibody.

Blood clotting suppresses titre in BAL liquid：Blood clotting suppresses titre average out to Log 2 in secondary vaccine Nasal immunization pig BAL liquid (79.5 ± 56.5), blood clotting suppresses titre average out in independent PRRSV-KPWV-SWIVA-RV antigens Nasal immunization pig BAL liquid Log 2 (42.1 ± 48.6), physiological saline simulate blood clotting in immune swine BAL liquid and suppress titre average out to Log 2 (4.7 ± 0.5), Blood clotting in malicious pig BAL liquid is only attacked unavoidably suppresses titre average out to Log 2 (23.2 ± 21.3).Secondary vaccine Nasal immunization pig BAL In liquid blood clotting suppress titre be significantly higher than physiological saline simulation immune swine BAL liquid in blood clotting suppress titre (P ＜ 0.001, also significantly Higher than in independent PRRSV-KPWV-SWIVA-RV antigens collunarium BAL liquid blood clotting suppress titre (P ＜ 0.01, also significantly greater than not Exempt from only to attack blood clotting suppression titre in malicious pig BAL liquid and (P ＜ 0.001, illustrate that the vaccine-induced pig of invention produces high-level pig and suppresses anti- Body, stable adjuvant compound can strengthen pig lung tissue and produce solidifying suppression antibody tormation.

The vaccine (the stable adjuvant compounds of PRRSKPWV-SwIVA-RV+) of the present invention of embodiment 6. is exempted to swine influenza virus Epidemic disease phase

6.1 experimental designs are collected with sample

Health, confirmation are without SwIAV H1N1 and H1N2 hemagglutination inhibition antibody piglets (n=60), and selected piglet immunological program is such as Embodiment 5, completed secondary Nasal immunization in one month, and attack time difference is divided into 4 test groups (10/groups of n=) and 4 pairs According to group (n=10 heads/group), packet such as table 5, the experimental animal welfare formulated by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences are done Method is raised and test operation.Sample is collected, virus titer detects, antibody titer detects, neutralizing antibody detects and statistical analysis By embodiment 6.

Table 5 is grouped situation

Different time attacks malicious humoral immune reaction after 6.2 Nasal immunizations

6.2.1 attack poison within 60 days after Nasal immunization, the specific system of pig, local immunity reaction are detected and analyzed.

Hog snout swab Specific IgA antibody：The average OD450nm of vaccine Nasal immunization hog snout swab Specific IgA antibody reaches It is 0.29 ± 0.3 that 1.4 ± 0.2, PBS Nasal immunization, which only attack the average OD450nm of malicious hog snout swab Specific IgA antibody,.Vaccine drips Nose immune swine nose swab Specific IgA antibody level is significantly higher than PBS simulation immune swine nose swab Specific IgA antibodies (P ＜ 0.001.After illustrating invention vaccine immunity 60 days, the high-caliber schneiderian membrane IgA antibody of H2N1 infection-induceds, antibody level with The antibody level of secondary vaccine immunity postoperative infection attack in 30 days is without significant difference.

IgA in lung lysate：IgA OD450nm average out to 1.50 ± 0.60 in vaccine Nasal immunization pig lung lysate, IgA OD450nm average out to 0.29 ± 0.65 in PBS simulation Immunization pig lung lysates.Vaccine Nasal immunization pig lung lysate Reclaimed water is flat to be significantly higher than IgA levels (P ＜ 0.001 in PBS simulation Immunization pig lung lysates.

BAL liquid IgA：Vaccine Nasal immunization pig BAL liquid IgA OD450nm average out to 2.0 ± 0.28, PBS simulations are immune to attack Malicious pig BAL liquid IgA OD450nm average out to 0.30 ± 0.18.Vaccine Nasal immunization pig BAL liquid IgA levels are significantly higher than PBS moulds It is horizontal (P ＜ 0.001) to intend Immunization pig BAL liquid IgA.

Swine plasma IgG antibody is reacted：Vaccine Nasal immunization Swine plasma specific IgG antibodies OD450nm average out to 0.49 ± 1.0, PBS simulation Immunization Swine plasma specific IgG antibodies OD450nm average out to 0.26 ± 0.48.Vaccine Nasal immunization pig Plasma specific IgG antibody level is significantly higher than PBS simulation Immunization Swine plasma specific IgG antibody levels ((P ＜ 0.01).Illustrate invention vaccine 60 days after immune, H1N2 attacks poison and reacted induction of systemic humoral immune.

BAL liquid IgG：Vaccine Nasal immunization attacks malicious pig BAL liquid IgG OD450nm average out to 0.80 ± 0.68, PBS simulations Immunization pig BAL liquid IgG OD450nm average out to 0.10 ± 0.3.Vaccine Nasal immunization pig BAL liquid IgG levels are significantly higher than PBS simulation Immunization pig BAL liquid IgG is horizontal (P ＜ 0.001), illustrates invention vaccine porcine mucosa local I gG after immune 60 days Level can anti-HN2 infection.

Blood clotting suppresses titre in BAL liquid：Vaccine Nasal immunization attacks blood clotting in malicious pig BAL liquid and suppresses titre average out to Log 2 (58.9 ± 40.2), PBS simulate blood clotting in Immunization pig BAL liquid and suppress titre average out to Log 2 (20.1 ± 14.6).Epidemic disease Seedling Nasal immunization pig attacks after poison blood clotting in BAL liquid and suppresses titre and be significantly higher than blood clotting in PBS simulation Immunization pig BAL liquid to suppress Titre (P ＜ 0.001), illustrates after invention vaccine immunity 60 days, still has enough antibody in pig lung, HIN2 infection excites high level Hemagglutination inhibition antibody.

Swine plasma blood clotting suppresses titre：Vaccine Nasal immunization attacks malicious Swine plasma blood clotting and suppresses titre average out to Log 2 (79.2 ± 50.1), PBS simulation Immunization Swine plasma blood clottings suppress titre average out to Log 2 (50.4 ± 1.8).Vaccine collunarium Immune Swine plasma blood clotting suppresses titre and is significantly higher than PBS simulation Immunization Swine plasma blood clotting suppression titres (P ＜ 0.001), says Bright invention vaccine immunity can maintain by 60 days induction of high-level Swine plasma hemagglutination inhibition antibody, height is excited after being infected by H1N2 Plasma levels hemagglutination inhibition antibody generates.

BAL liquid neutralizing antibodies：Vaccine immunity attacks malicious pig BAL liquid neutralizing antibody average out to Log 2 (76.9 ± 19.3), PBS Simulate Immunization pig BAL liquid neutralizing antibody average out to Log 2 (21.2 ± 19.3).Vaccine immunity pig BAL liquid neutralizing antibody water It is flat to be significantly higher than PBS simulation Immunization pig BAL liquid neutralizing antibody levels (P ＜ 0.001).Illustrate that invention vaccine exempts from latter 60 days still There is higher level neutralizing antibody, H1N2 infection attacks still induce pig lung tissue to produce high-level neutralizing antibody.

6.2.2 90 days after Nasal immunization, H1N1 attacks poison, and pig specific system, local immunity reaction are detected and divided Analysis.

Hog snout swab Specific IgA antibody：Vaccine immunity hog snout swab Specific IgA antibody OD450nm average 0.95 ± 0.32, PBS Nasal immunization attacks malicious hog snout swab Specific IgA antibody OD450nm average out to 0.16 ± 0.19.Vaccine immunity hog snout Swab Specific IgA antibody level is significantly higher than PBS simulation immune swine nose swab Specific IgA antibodies (P ＜ 0.001).Explanation Invention vaccine immunity pig body still protective IgA antibody after 90 days, the high-caliber schneiderian membrane IgA antibody of H1N1 infection-induceds, The antibody level of antibody level and vaccine immunity postoperative infection attack in 60 days is without significant difference.

IgA in lung lysate：IgA OD450nm average out to 0.84 ± 0.41 in vaccine Nasal immunization pig lung lysate, IgA OD450nm average out to 0.34 ± 0.51 in PBS simulation Immunization pig lung lysates.Vaccine Nasal immunization pig lung lysate Reclaimed water is flat to be significantly higher than IgA levels (P ＜ 0.01) in PBS simulation Immunization pig lung lysates.

BAL liquid IgA：Vaccine Nasal immunization pig BAL liquid IgA OD450nm average out to 1.2 ± 0.11, PBS simulations are immune to attack Malicious pig BAL liquid IgA OD450nm average out to 0.14 ± 0.15.Vaccine Nasal immunization pig BAL liquid IgA levels are significantly higher than PBS moulds It is horizontal (P ＜ 0.001) to intend Immunization pig BAL liquid IgA.

Swine plasma IgG antibody is reacted：Vaccine immunity Swine plasma specific IgG antibodies OD450nm average out to 2.3 ± 0.86, PBS simulation Immunization Swine plasma specific IgG antibodies OD450nm average out to 0.17 ± 0.23.Vaccine immunity Swine plasma is special Property IgG antibody level to be significantly higher than PBS simulation Immunization Swine plasma specific IgG antibodies horizontal (P ＜ 0.01).Illustrate hair Bright vaccine still has the systemic humoral protective immunological reaction of anti-H1N1 infection 90 days after immune.

BAL liquid IgG：Vaccine immunity attacks malicious pig BAL liquid IgG OD450nm average out to 0.69 ± 0.42, and PBS simulations are immune to attack Malicious pig BAL liquid IgG OD450nm average out to 0.23 ± 0.4.Vaccine immunity pig BAL liquid IgG levels are significantly higher than PBS simulations and exempted from Epidemic disease attacks malicious pig BAL liquid IgG levels and (P ＜ 0.01, illustrates that invention vaccine keeps mucous membrane local I gG horizontal anti-after immune 90 days H1N1 infects.

Blood clotting suppresses titre in BAL liquid：Vaccine immunity attacks blood clotting in malicious pig BAL liquid and suppresses titre average out to Log 2 (41.5 ± 20.5), PBS simulate blood clotting in Immunization pig BAL liquid and suppress titre average out to Log 2 (15.3 ± 8.4).Vaccine Immune swine attacks after poison blood clotting in BAL liquid and suppresses titre and be significantly higher than blood clotting in PBS simulation Immunization pig BAL liquid to suppress titre (P ＜ 0.01), illustrate after invention vaccine immunity still there is enough antibody, the high-level anti-HIN1 of hemagglutination inhibition antibody in pig lung 90 days Infection.

Swine plasma blood clotting suppresses titre：Vaccine immunity attack malicious Swine plasma blood clotting suppress titre average out to Log 2 (46.6 ± 20.3), PBS simulates Immunization Swine plasma blood clotting suppression titre average out to Log 2 (10.3 ± 1.5).Vaccine immunity Swine plasma Blood clotting suppresses titre and is significantly higher than PBS simulation Immunization Swine plasma blood clotting suppression titres (P ＜ 0.001), illustrates invention vaccine It is immune, protectiveness hemagglutination inhibition antibody, it can be maintained in pig blood by 90 days, anti-H1N1 infection.

BAL liquid neutralizing antibodies：Vaccine immunity attacks malicious pig BAL liquid neutralizing antibody average out to Log 2 (33.5 ± 5.5), PBS Simulate Immunization pig BAL liquid neutralizing antibody average out to Log 2 (9.0 ± 6.5).Vaccine immunity pig BAL liquid neutralizing antibody is horizontal It is horizontal (P ＜ 0.01) to be significantly higher than PBS simulation Immunization pig BAL liquid neutralizing antibodies.Illustrate that invention vaccine exempts from latter 90 days in lung Tissue maintains the anti-H1N1 infection of higher level neutralizing antibody.

6.2.3 120 days after Nasal immunization, H1N2 attacks poison, to pig specific system, local immunity reaction detected and Analysis.

Hog snout swab Specific IgA antibody：Vaccine immunity hog snout swab Specific IgA antibody OD450nm average out tos 0.8 ± 0.2, PBS immune only attacks malicious hog snout swab Specific IgA antibody OD450nm average out to 0.26 ± 0.2.Vaccine immunity hog snout swab Specific IgA antibody level is significantly higher than PBS simulation immune swine nose swab Specific IgA antibodies (P ＜ 0.01).Illustrate invention After vaccine immunity 120 days, schneiderian membrane maintains high-level IgA antibody effectively to protect H1N2 to infect.

IgA in lung lysate：IgA OD450nm average out to 0.65 ± 0.34 in vaccine immunity pig lung lysate, PBS moulds Intend IgA OD450nm average out to 0.24 ± 0.35 in Immunization pig lung lysate.IgA in vaccine Nasal immunization pig lung lysate Level is significantly higher than IgA levels (P ＜ 0.01 in PBS simulation Immunization pig lung lysates.

BAL liquid IgA：Vaccine immunity pig BAL liquid IgA OD450nm average out to 0.81 ± 0.22, PBS simulation Immunization pigs BAL liquid IgA OD450nm average out to 0.35 ± 0.21.Vaccine Nasal immunization pig BAL liquid IgA levels are significantly higher than PBS simulations and exempted from It is horizontal (P ＜ 0.01) that epidemic disease attacks malicious pig IgA.

Swine plasma IgG antibody is reacted：Vaccine immunity Swine plasma specific IgG antibodies OD450nm average out to 0.85 ± 0.61, PBS simulation Immunization Swine plasma specific IgG antibodies OD450nm average out to 0.29 ± 0.48.Vaccine Nasal immunization pig Plasma specific IgG antibody level is significantly higher than PBS simulation Immunization Swine plasma specific IgG antibodies levels (P ＜ 0.01).Illustrate invention vaccine immunity pig Swine plasma still protective anti-igg antibody, anti-H1N2 infection after 120 days.

BAL liquid IgG：Vaccine immunity pig BAL liquid IgG OD450nm average out to 0.47 ± 0.31, PBS simulation Immunization pigs BAL liquid IgG OD450nm average out to 0.13 ± 0.21.Vaccine Nasal immunization pig BAL liquid IgG levels are significantly higher than PBS simulations and exempted from Epidemic disease attacks malicious pig BAL liquid IgG levels (P ＜ 0.01), illustrates that invention vaccine porcine mucosa local I gG levels after immune 120 days can resist HN2 infects.

Blood clotting suppresses titre in BAL liquid：Vaccine immunity attacks blood clotting in malicious pig BAL liquid and suppresses titre average out to Log 2 (33.6 ± 13.5), PBS simulate blood clotting in Immunization pig BAL liquid and suppress titre average out to Log 2 (21.1 ± 11.7).Vaccine Immune swine attacks after poison blood clotting in BAL liquid and suppresses titre and be significantly higher than blood clotting in PBS simulation Immunization pig BAL liquid to suppress titre (P ＜ 0.01), illustrate after invention vaccine immunity still there is enough hemagglutination inhibition antibodies, anti-HIN2 infection in pig lung 120 days.

Swine plasma blood clotting suppresses titre：Vaccine immunity Swine plasma blood clotting suppresses titre average out to Log 2 (46.8 ± 32.1), PBS simulation Immunization Swine plasma blood clottings suppress titre average out to Log 2 (33.7 ± 2.7).Vaccine immunity Swine plasma blood clotting presses down Titre processed is significantly higher than PBS simulation Immunization Swine plasma blood clottings and suppresses titre (P ＜ 0.01), illustrates that invention vaccine immunity induces High-level Swine plasma hemagglutination inhibition antibody is simultaneously maintained by 120 days, and anti-H1N2 is in pulmonary infection.

BAL liquid neutralizing antibodies：Vaccine immunity pig BAL liquid neutralizing antibody average out to Log 2 (34.5 ± 9.7), PBS are simulated Immunization pig BAL liquid neutralizing antibody average out to Log 2 (20.9 ± 12.6).Vaccine immunity pig BAL liquid neutralizing antibody is horizontal aobvious Write horizontal (P ＜ 0.01) higher than PBS simulation Immunization pig BAL liquid neutralizing antibodies.Illustrate invention vaccine Mian Hou120Tian pigs lung Still there is the anti-H1N2 infection attack of higher level neutralizing antibody.

6.2.4 150 days after Nasal immunization, H1N2 attacks poison, to pig specific system, local immunity reaction detected and Analysis.

Hog snout swab Specific IgA antibody：Vaccine immunity hog snout swab Specific IgA antibody OD450nm average out tos 0.31 ± 0.3, PBS simulation Immunization hog snout swab Specific IgA antibody OD450nm average out to 0.24 ± 0.21.Vaccine immunity pig Nose swab Specific IgA antibody is horizontal to simulate immune swine nose swab Specific IgA antibody level without significant difference (P ＜ with PBS 0.05).After illustrating vaccine immunity 150 days, hog snout mucosal IgA antibodies level can not effectively protect lung caused by H1N2 infection attacks Portion's damage, pathology and clinical respiratory disorder symptom.

IgA in lung lysate：IgA OD450nm average out to 0.31 ± 0.34 in vaccine immunity pig lung lysate, PBS moulds Intend IgA OD450nm average out to 0.27 ± 0.25 in Immunization pig lung lysate.IgA in vaccine Nasal immunization pig lung lysate IgA levels are without significant difference (P ＜ 0.05) in horizontal and PBS simulation Immunization pig lung lysates.Illustrate vaccine immunity 150 days Afterwards, pig pneumonocyte, which produces IgA antibody level, can not effectively protect pulmonary lesion, pathology and clinic caused by H1N2 infection attacks to exhale Inhale disease symptomses.

BAL liquid IgA：Vaccine immunity pig BAL liquid IgA OD450nm average out to 0.36 ± 0.15, PBS simulation Immunization pigs BAL liquid IgA OD450nm average out to 0.37 ± 0.11.Vaccine Nasal immunization pig BAL liquid IgA is horizontal and PBS simulates Immunization Pig IgA levels are without significant difference (P ＜ 0.05).

Swine plasma IgG antibody is reacted：Vaccine immunity Swine plasma specific IgG antibodies OD450nm average out to 0.32 ± 0.34, PBS simulation Immunization Swine plasma specific IgG antibodies OD450nm average out to 0.31 ± 0.21.Vaccine Nasal immunization pig Plasma specific IgG antibody is horizontal to simulate the horizontal indifference of Immunization Swine plasma specific IgG antibodies ((P ＜ with PBS 0.05).After illustrating vaccine immunity pig 120 days, Swine plasma protectiveness anti-igg antibody, which has declined, can not resist H1N2 infection.

BAL liquid IgG：Vaccine immunity pig BAL liquid IgG OD450nm average out to 0.20 ± 0.19, PBS simulation Immunization pigs BAL liquid IgG OD450nm average out to 0.18 ± 0.13.Vaccine Nasal immunization pig BAL liquid IgG is horizontal to simulate Immunization with PBS The horizontal indifferences of pig BAL liquid IgG (P ＜ 0.05), illustrate invention vaccine immunity pig after 150 days, porcine mucosa local I gG levels are HN2 infection is not resistant to.

Blood clotting suppresses titre in BAL liquid：Vaccine immunity attacks blood clotting in malicious pig BAL liquid and suppresses titre average out to Log 2 (22.3 ± 9.4), PBS simulate blood clotting in Immunization pig BAL liquid and suppress titre average out to Log 2 (24.1 ± 7.6).Vaccine is exempted from Epidemic disease pig attacks after poison blood clotting in BAL liquid and suppresses titre suppresses titre without significant difference with blood clotting in PBS simulation Immunization pig BAL liquid (P ＜ 0.05), illustrate that hemagglutination inhibition antibody disappeared through 150 days in invention vaccine immunity induction lung branch bronchoalveolar, Pig lung without enough hemagglutination inhibition antibodies, resists anti-HIN2 infection.

Swine plasma blood clotting suppresses titre：Vaccine immunity Swine plasma blood clotting suppresses titre average out to Log 2 (29.5 ± 11.0), PBS simulation Immunization Swine plasma blood clottings suppress titre average out to Log 2 (26.7 ± 10.1).Vaccine immunity Swine plasma blood clotting presses down Titre processed suppresses titre without significant difference (P ＜ 0.0) with PBS simulation Immunization Swine plasma blood clottings.Illustrate invention vaccine immunity Induction of pig high level blood plasma hemagglutination inhibition antibody and maintain by 120 days, disappeared within 150 days, in pig lung without enough Hemagglutination inhibition antibody, resist anti-HIN2 infection.

BAL liquid neutralizing antibodies：Vaccine immunity pig BAL liquid neutralizing antibody average out to Log 2 (22.3 ± 4.3), PBS are simulated Immunization pig BAL liquid neutralizing antibody average out to Log 2 (23.2 ± 5.1).Vaccine immunity pig BAL liquid neutralizing antibody it is horizontal and PBS simulates Immunization pig BAL liquid neutralizing antibody levels and illustrates that invention vaccine exempts from rear 150 days pigs without significant difference (P ＜ 0.05) Lung is without the anti-H1N2 infection attack of high-level neutralizing antibody.

To sum up, the secondary Nasal immunization of the vaccine, duration of immunity are 120 days.

Claims

1. a kind of porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virosomes vaccine, it is characterised in that numerous containing pig Grow and reconstruct subvirion antigens with breathing syndrome virus-swine influenza virus and stablize adjuvant compound, described pig is bred with exhaling It is by directly adsorbing or being total to by porcine reproductive and respiratory syndrome virus to inhale syndrome virus-swine influenza virus reconstruct virion Valency is incorporated in swine influenza virus reconstruct virosomal surface and formed, wherein, described swine influenza virus reconstruct virion is containing phosphorus The vesicles of lipid bilayer, be combined with the surface of phospholipid bilayer swine influenza virus hemagglutinin (haemagglutinin, ) and neuraminidase (neuraminidase, NA) albumen HA.

2. porcine reproductive and respiratory syndrome virus as claimed in claim 1-swine influenza virus reconstruct virosomes vaccine, its feature It is, described stable adjuvant compound is must be more with non-essential amino acid blend, 20~25g/L containing 2.0~10g/L Polylysine hydroxymethyl cellulose compound (Poly ICLC), 0.1-0.5mg/L BCG vaccines lysate, 30~100g/L disaccharide, 30~100g/L polyalcohols, 1.0~5.0g/L urea or urea derivative, 0.01~0.4g/L EDTA or edta salt, 0.001 10~100mM of~0.1g/L surfactants cushioning liquid, preferably pH value 7.0~9.0, pH 7.2-8.0.

3. porcine reproductive and respiratory syndrome virus as claimed in claim 2-swine influenza virus reconstruct virosomes vaccine, its feature It is, described must comprise at least arginine or arginine salt and glutamic acid or glutamic acid with non-essential amino acid blend Salt；Described disaccharide is non-animal source disaccharide, preferably maltose, fucose or sucrose wherein at least one, and described is polynary Alcohol is non-animal source polyalcohol, preferably D-sorbite or mannitol or its combination, and described surfactant is nonionic Surfactant, preferably Tween-20 or Pluronic F68；Described cushioning liquid is selected from phosphate buffer, Tris buffer solutions Or the mixture that any one or more than one of HEPES buffer solution are formed in arbitrary volume ratio.

4. porcine reproductive and respiratory syndrome virus as claimed in claim 2-swine influenza virus reconstruct virosomes vaccine, its feature It is, described BCG vaccine lysate is prepared in accordance with the following methods：BCG vaccine improvement Soviet Union 37 DEG C of static trainings of logical synthetic medium Support 2-3 weeks, culture carries out sterilization processing in 30 minutes at 121 DEG C, and thalline, PBS washings 2 are harvested with preparative low speed centrifuge It is secondary, be suspended in containing EDTA, protease inhibitors, DNA enzymatic, RNase PBS in, bacterium is crushed with bead, until 90% bacterium Body crushes, the unbroken cell of centrifugation, insoluble cell wall constituent, harvest cracking supernatant；Supernatant through 0.2 μM, it is low Protein bound membrane filtration, obtains whole cell lysate, and wherein level of endotoxin should be not higher than 0.002 μ g/mg albumen.

5. porcine reproductive and respiratory syndrome virus as claimed in claim 1-swine influenza virus reconstruct virosomes vaccine, its feature It is, in described vaccine, porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct subvirion antigens content is 4-8 μ g/ ml。

A kind of 6. porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct prepared as described in claim any one of 1-5 Virosomes vaccine method, it is characterised in that comprise the following steps：

(1) preparation of the PRRSV totivirus antigens of purifying inactivation；

(3) preparation of swine influenza virus reconstruct virion

The preparation of a phospholipid dispersions

Prepared in homogenizer with the 0.01M Tris/HCl containing pH 7.3,0.1M NaCl and mixed containing phosphatide and cholesterol The dispersion liquid of thing, wherein containing 75% phosphatidyl courage in percentage by weight in described phosphatide and the mixture of cholesterol Alkali, 20% phosphatidyl-ethanolamine and 5% cholesterol, wherein all phosphatide account for the 1-2% (w/v) of dispersion liquid, by cholic acid Sodium is added in dispersion liquid, makes its final concentration of 1.3% (w/v)；

The preparation of b swine influenza virus outer membrane proteins

Added into the swine influenza virus virus liquid of purifying containing the glycol list dodecyl ethers of 0.1M eight, 7.9mg/ml NaCl, The pH 7.3 of 4.4mg/ml sodium citrates, 2.1mg/ml MES, 1.2mg/ml N- ethoxys-piperazine-N'-2- ethyl sulfonic acids water Solution, mixture centrifuge in ultracentrifuge, collect supernatant, obtain viral envelope proteins, i.e. hemagglutinin (HA) and neural ammonia Sour enzyme (NA)；

C swine influenza viruses reconstruct the preparation of virion

The supernatant that step b is obtained is added in the phospholipid dispersions that step a is obtained, in 4 DEG C of stirrings, loading Sephadex G- 50 chromatographic columns, pillar is placed to be connected with Ultrasound Instrument in a water bath, and ultrasonic vibration per minute produces swine influenza virus reconstruct disease for 10 seconds Malicious body, reconstituted influenza virion and cholesterol micro-capsule flow out in outer water section, collect void volume part, will contain swine influenza virus The outer hydration of reconstructed volume simultaneously, chromatographs again under similarity condition, obtains swine influenza virus reconstructed volume；

The swine influenza virus reconstructed volume that the PRRSV totivirus antigen and step (2) that the inactivation that step (1) is obtained purifies obtain is mixed Close, gently shake resuspension, being gently mixed at 20 DEG C makes PRRSV be adsorbed in swine influenza virus reconstruct disease by Van der Waals force for 48 hours The surface of malicious body, obtain porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virion；Or

Disulfide bond be present using PRRSV surfaces and carried out covalent bond with swine influenza virus reconstruct virion.

A kind of 7. porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct prepared as described in claim any one of 1-5 Virosomes vaccine method, it is characterised in that comprise the following steps：

(1) preparation of the PRRSV totivirus antigens of purifying inactivation

(2) PRRSV's is Thiolation

The PRRSV of inactivation purifying is dissolved in 0.1M phosphate buffers；N- succinimido pyridine radicals dithiopropionic acid esters (SPDP) mixed with ethanol, take mixed liquor to be slowly added to the phosphate buffer containing PRRSV with syringe, make SPDP and PRRSV Mol ratio 15:1, while keep concentration of alcohol to prevent albuminous degeneration in below 5v/v%, mixture reacts 30 minutes at 20 DEG C, After reaction terminates, put down respectively with pH7.0 0.05M sodium citrates, pH7.0 0.05M sodium phosphates, pH7.0 0.05M sodium chloride Weighing apparatus Sephadex G-50 pillar is purified three times；Obtain Thiolation PRRSV totivirus antigens PRRSV-SPDP；

(4) preparation of swine influenza virus reconstruct virion

The preparation of a phospholipid dispersions

Phosphatidyl choline (PE) and N- succinimido pyridine radicals dithiopropionic acid esters (SPDP) are crosslinked：15mg phosphatide Phatidylcholine be put into 5ml vials drying, dry after be redissolved in chloroform, then add containing triethylamine (TEA), SPDP it is anhydrous 1-2 hours are stirred at room temperature until reaction is completed in ethanol, mixture under logical condition of nitrogen gas, i.e., no single phosphatidyl choline, instead Product is answered to dry on a rotary evaporator, product is resuspended in chloroform, and loading silicic acid chromatography post is purified, and obtains PE-SPDP；

Prepared in homogenizer with the 0.01M Tris/HCl containing pH 7.3,0.1M NaCl and mixed containing phosphatide and cholesterol The dispersion liquid of thing, wherein containing 75% PE- in percentage by weight in described phosphatide and the mixture of cholesterol SPDP, 20% phosphatidyl-ethanolamine and 5% cholesterol, wherein all phosphatide account for the 1-2% (w/v) of dispersion liquid, by courage Sour sodium is added in phospholipid dispersions, makes its final concentration of 1.3% (w/v)；

The preparation of b swine influenza virus outer membrane proteins

Added into the swine influenza virus virus liquid of purifying containing the glycol list dodecyl ethers of 0.1M eight, 7.9mg/mlNaCl, The pH 7.3 of 4.4mg/ml sodium citrates, 2.1mg/ml MES, 1.2mg/ml N- ethoxys-piperazine-N'-2- ethyl sulfonic acids water Solution, mixture centrifuge in ultracentrifuge, collect supernatant, obtain viral envelope proteins, i.e. hemagglutinin (HA) and neural ammonia Sour enzyme (NA)；

C swine influenza viruses reconstruct the preparation of virion

Thiolation PRRSV totivirus antigen PRRSV-SPDP pH is adjusted with 1M HCl in 0.2M citrate phosphate buffers To 5.5, dithiothreitol (DTT) solution is added, solution is placed 30 minutes, and balancing Sephadex G-50 with PBS separates two sulphur Threitol, albumen is collected in having nitrogen environment, the Thiolation PRRSV totivirus antigen after being reduced；By the reconstruct of acquisition Thiolation PRRSV totivirus antigen after swine influenza virus body and reduction is stirred at room temperature overnight, and obtains pig breeding and breathing Syndrome virus-swine influenza virus reconstituted virosomes.

8. method as claimed in claims 6 or 7, it is characterised in that the system of the PRRSV totivirus antigens of described purifying inactivation It is standby to comprise the following steps：

(1) Virus culture, three times freeze-thaw, harvest

Porcine reproductive and respiratory syndrome virus infection MARC-145 cells, harvest virus-culturing fluid, collect PRRSV virus liquids through three Secondary freeze-thaw, lysate centrifugation, supernatant collection is in sterile chamber tank；

(2) inactivate

(3) ion-exchange chromatography

Virus liquid after inactivation is chromatographed with ion exchange column；

(4) it is concentrated by ultrafiltration, dialyses

With cutoff value it is that 300KD films are concentrated by ultrafiltration from the virus liquid of ion exchange column elution, while with 0.02M Tris- HCl (pH 7.5) dialyses；

(5) molecular exclusion chromatography

(6) it is concentrated by ultrafiltration, dialyses

Viral purification liquid is used into film of the cutoff value for 300KDa, delayed using the 50mM phosphoric acid containing 150mM NaCl, pH 7.5 Fliud flushing ultrafiltration dialysis, obtain inactivation purifying PRRSV totivirus liquid.

9. method as claimed in claims 6 or 7, it is characterised in that the swine influenza virus totivirus antigen of described purifying inactivation Preparation comprise the following steps：

(1) Virus culture, three times freeze-thaw, harvest

Swine influenza virus infection mdck cell, virus-culturing fluid is harvested, collect swine influenza virus virus liquid through freeze-thaw three times, cracking Liquid centrifuges, and supernatant collection is in sterile chamber tank；

(2) inactivate

(3) ion-exchange chromatography

Virus liquid after inactivation is chromatographed with ion exchange column；

(4) it is concentrated by ultrafiltration, dialyses

With cutoff value it is that 700KD films are concentrated by ultrafiltration from the virus liquid of ion exchange column elution, while with 0.02M Tris- HCl (pH 7.5) dialyses；

(5) molecular exclusion chromatography

(6) it is concentrated by ultrafiltration, dialyses

Viral purification liquid is used into film of the cutoff value for 700KDa, delayed using the 50mM phosphoric acid containing 150mM NaCl, pH 7.5 Fliud flushing ultrafiltration dialysis, obtain inactivation purifying swine influenza virus totivirus liquid.

10. porcine reproductive and respiratory syndrome virus-swine influenza virus reconstruct virosomes vaccine described in claim any one of 1-5 Purposes in medicine of the preparation prevention by the disease caused by porcine reproductive and respiratory syndrome virus and/or swine influenza virus, Preferably, described medicine is administered by way of collunarium.